Method for producing sweetners and alcohol

ABSTRACT

The present invention relates to variants of a parent α-amylase, which parent α-amylase (i) has an amino acid sequence selected from the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 7, respectively; or (ii) displays at least 80% homology with one or more of these amino acid sequences; and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of these amino acid sequences; and/or is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of these amino acid sequences; in which variant:  
     (a) at least one amino acid residue of the parent α-amylase has been deleted; and/or  
     (b) at least one amino acid residue of the parent α-amylase has been replaced by a different amino acid residue; and/or  
     (c) at least one amino acid residue has been inserted relative to the parent α-amylase; the variant having α-amylase activity and exhibiting at least one of the following properties relative to the parent α-amylase: increased thermostability; increased stability towards oxidation; and reduced Ca 2+  dependency; with the proviso that the amino acid sequence of the variant is not identical to any of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No.10/025,648, filed Dec. 19, 2001, which is a division of U.S. patentapplication Ser. No. 09/902,188, filed on Jul. 10, 2001, which is acontinuation of U.S. patent application Ser. No. 09/354,191, now U.S.Pat. No. 6,297,038, filed on Jul. 15, 1999, which is a continuation ofU.S. patent application Ser. No. 08/600,656, now U.S. Pat. No.6,093,562, filed on Feb. 13, 1996, which is a continuation ofapplication serial no. PCT/DK96/00056, filed on Feb. 5, 1996, whichclaims priority under 35 U.S.C. 119 of Danish application serial nos.0126/95, filed on Feb. 3, 1995, 0336/95, filed on Mar. 29, 1995,1097/95, filed on Sep. 29, 1995, and 1121/95, filed on Oct. 6, 1995, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to α-amylase variants havingimproved properties relative to the parent enzyme (e.g. improved thermaland/or oxidation stability and/or reduced calcium ion dependency), andthereby improved washing and/or dishwashing (and/or textile desizing)performance. The invention also relates to DNA constructs encoding thevariants, and to vectors and cells harboring the DNA constructs. Theinvention further relates to methods of producing the amylase variants,and to detergent additives and detergent compositions comprising theamylase variants. Furthermore, the invention relates to the use of theamylase variants for textile desizing.

BACKGROUND OF THE INVENTION

[0003] α-Amylase enzymes have been used industrially for a number ofyears and for a variety of different purposes, the most important ofwhich are starch liquefaction, textile desizing, starch modification inthe paper and pulp industry, and for brewing and baking. A further useof α-amylases which is becoming increasingly important is the removal ofstarchy stains during washing or dishwashing.

[0004] In recent years attempts have been made to construct α-amylasevariants having improved properties with respect to specific uses suchas starch liquefaction and textile desizing.

[0005] For instance, U.S. Pat. No. 5,093,257 discloses chimericα-amylases comprising an N-terminal part of a B. stearothermophilusα-amylase and a C-terminal part of a B. licheniformis α-amylase. Thechimeric α-amylases are stated to have unique properties, such as adifferent thermostability, as compared to their parent α-amylase.However, all of the specifically described chimeric α-amylases wereshown to have a decreased enzymatic activity as compared to their parentα-amylases.

[0006] EP 252 666 describes hybrid amylases of the general formulaQ-R-L, in which Q is a N-terminal polypeptide residue of from 55 to 60amino acid residues which is at least 75% homologous to the 57N-terminal amino acid residues of a specified α-amylase from B.amyloliquefaciens, R is a specified polypeptide, and L is a C-terminalpolypeptide comprising from 390 to 400 amino acid residues which is atleast 75% homologous to the 395 C-terminal amino acid residues of aspecified B. licheniformis α-amylase.

[0007] Suzuki et al. (1989) disclose chimeric α-amylases, in whichspecified regions of a B. amyloliquefaciens α-amylase have beensubstituted for the corresponding regions of a B. licheniformisα-amylase. The chimeric α-amylases were constructed with the purpose ofidentifying regions responsible for thermostability. Such regions werefound to include amino acid residues 177-186 and amino acid residues255-270 of the B. amyloliquefaciens α-amylase. The alterations of aminoacid residues in the chimeric α-amylases did not seem to affectproperties of the enzymes other than their thermostability.

[0008] WO 91/00353 discloses α-amylase mutants which differ from theirparent α-amylase in at least one amino acid residue. The α-amylasemutants disclosed in said patent application are stated to exhibitimproved properties for application in the degradation of starch and/ortextile desizing due to their amino acid substitutions. Some of themutants exhibit improved stability, but no improvements in enzymaticactivity were reported or indicated. The only mutants exemplified areprepared from a parent B. licheniformis α-amylase and carry one of thefollowing mutations: H133Y or H133Y+T149I. Another suggested mutation isA111T.

[0009] FR 2,676,456 discloses mutants of the B. licheniformis α-amylase,in which an amino acid residue in the proximity of His 133 and/or anamino acid residue in the proximity of Ala 209 have been replaced by amore hydrophobic amino acid residue. The resulting α-amylase mutants arestated to have an improved thermostability and to be useful in thetextile, paper, brewing and starch liquefaction industry.

[0010] EP 285 123 discloses a method of performing random mutagenesis ofa nucleotide sequence. As an example of such sequence a nucleotidesequence encoding a B. stearothermophilus α-amylase is mentioned. Whenmutated, an α-amylase variant having improved activity at low pH valuesis obtained.

[0011] In none of the above references is it mentioned or even suggestedthat α-amylase mutants may be constructed which have improved propertieswith respect to the detergent industry.

[0012] EP 525 610 relates to mutant enzymes having improved stabilitytowards ionic tensides (surfactants). The mutant enzymes have beenproduced by replacing an amino acid residue in the surface part of theparent enzyme with another amino acid residue. The only mutant enzymespecifically described in EP 525 610 is a protease. Amylase is mentionedas an example of an enzyme which may obtain an improved stabilitytowards ionic tensides, but the type of amylase, its origin or specificmutations are not specified.

[0013] WO 94/02597 discloses α-amylase mutants which exhibit improvedstability and activity in the presence of oxidizing agents. In themutant α-amylases, one or more methionine residues have been replacedwith amino acid residues different from Cys and Met. The α-amylasemutants are stated to be useful as detergent and/or dishwashingadditives as well as for textile desizing.

[0014] WO 94/18314 discloses oxidatively stable α-amylase mutants,including mutations in the M197 position of B. licheniformis α-amylase.

[0015] EP 368 341 describes the use of pullulanase and other amylolyticenzymes optionally in combination with an α-amylase for washing anddishwashing.

[0016] An object of the present invention is to provide α-amylasevariants which—relative to their parent α-amylase—possess improvedproperties of importance, inter alia, in relation to the washing and/ordishwashing performance of the variants in question, e.g. increasedthermal stability, increased stability towards oxidation, reduceddependency on Ca²⁺ ion and/or improved stability or activity in the pHregion of relevance in, e.g., laundry washing or dishwashing. Suchvariant α-amylases have the advantage, among others, that they may beemployed in a lower dosage than their parent α-amylase. Furthermore, theα-amylase variants may be able to remove starchy stains which cannot, orcan only with difficulty, be removed by α-amylase detergent enzymesknown today.

BRIEF DISCLOSURE OF THE INVENTION

[0017] A goal of the work underlying the present invention was toimprove, if possible, the stability of, inter alia, particularα-amylases which are obtainable from Bacillus strains and whichthemselves had been selected on the basis of their starch removalperformance in alkaline media (such as in detergent solutions astypically employed in laundry washing or dishwashing) relative to manyof the currently commercially available α-amylases. In this connection,the present inventors have surprisingly found that it is in factpossible to improve properties of the types mentioned earlier (videsupra) of such a parent α-amylase by judicial modification of one ormore amino acid residues in various regions of the amino acid sequenceof the parent α-amylase. The present invention is based on this finding.

[0018] Accordingly, in a first aspect the present invention relates tovariants of a parent α-amylase, the parent α-amylase in question beingone which:

[0019] i) has one of the amino acid sequences shown in SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively, herein; or

[0020] ii) displays at least 80% homology with one or more of the aminoacid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQID No. 7; and/or displays immunological cross-reactivity with anantibody raised against an α-amylase having one of the amino acidsequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ IDNo. 7, respectively; and/or is encoded by a DNA sequence whichhybridizes with the same probe as a DNA sequence encoding an α-amylasehaving one of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3 and SEQ ID No. 7, respectively.

[0021] An α-amylase variant of the invention is subject to the provisothat it is a variant which does not have an amino acid sequenceidentical to the amino acid sequence shown in SEQ ID No. 1, in SEQ IDNo. 2, in SEQ ID No. 3 or in SEQ ID No. 7.

[0022] DNA sequences encoding the first three of the α-amylase aminoacid sequences in question are shown in SEQ ID No. 4 (encoding the aminoacid sequence shown in SEQ ID No. 1), SEQ ID No. 5 (encoding the aminoacid sequence shown in SEQ ID No. 2) and SEQ ID No. 6 (encoding theamino acid sequence shown in SEQ ID No. 3).

[0023] The amino acid sequences of the SEQ ID No. 1 and SEQ ID No. 2parent α-amylases, and the corresponding DNA sequences (SEQ ID No. 4 andSEQ ID No. 5, respectively) are also disclosed in WO 95/26397 (under thesame SEQ ID Nos. as in the present application).

[0024] The variants of the invention are variants in which: (a) at leastone amino acid residue of the parent α-amylase has been deleted; and/or(b) at least one amino acid residue of the parent α-amylase has beenreplaced (i.e. substituted) by a different amino acid residue; and/or(c) at least one amino acid residue has been inserted relative to theparent α-amylase. The variants in question have themselves α-amylaseactivity and exhibit at least one of the following properties relativeto the parent α-amylase:

[0025] increased thermostability, i.e. satisfactory retention ofenzymatic activity at a temperature higher than that suitable for usewith the parent enzyme;

[0026] increased oxidation stability, i.e. increased resistance todegradation by oxidants (such as oxygen, oxidizing bleaching agents andthe like);

[0027] reduced Ca²⁺ dependency, i.e. the ability to functionsatisfactorily in the presence of a lower Ca²⁺ concentration than in thecase of the parent α-amylase. α-Amylases with such reduced Ca²⁺dependency are highly desirable for use in detergent compositions, sincesuch compositions typically contain relatively large amounts ofsubstances (such as phosphates, EDTA and the like) which bind calciumions strongly.

[0028] Examples of other desirable improvements or modifications ofproperties (relative to the parent α-amylase in question) which may beachieved with a variant according to the invention are:

[0029] increased stability and/or α-amylolytic activity at neutral torelatively high pH values, e.g. at pH values in the range of 7-10.5,such as in the range of 8.5-10.5;

[0030] increased α-amylolytic activity at relatively high temperatures,e.g. temperatures in the range of 40-70° C.;

[0031] increase or decrease of the isoelectric point (pI) so as tobetter match the pI value for the α-amylase variant in question to thepH of the medium (e.g. a laundry washing medium, dishwashing medium ortextile-desizing medium) in which the variant is to be employed (videinfra); and

[0032] improved binding of a particular type of substrate, improvedspecificity towards a substrate, and/or improved specificity withrespect to cleavage (hydrolysis) of substrate.

[0033] An amino acid sequence is considered to be X % homologous to theparent α-amylase if a comparison of the respective amino acid sequences,performed via known algorithms, such as the one described by Lipman andPearson in Science 227 (1985) p. 1435, reveals an identity of X %. TheGAP computer program from the GCG package, version 7.3 (June 1993), maysuitably be used, employing default values for GAP penalties [GeneticComputer Group (1991) Programme Manual for the GCG Package, version 7,575 Science Drive, Madison, Wis., USA 53711].

[0034] In the context of the present invention, “improved performance”as used in connection with washing and dishwashing is, as alreadyindicated above, intended to mean improved removal of starchy stains,i.e. stains containing starch, during washing or dishwashing,respectively. The performance may be determined in conventional washingand dishwashing experiments and the improvement evaluated as acomparison with the performance of the parent α-amylase in question. Anexample of a small-scale “mini dishwashing test” which can be used anindicator of dishwashing performance is described in the Experimentalsection, below.

[0035] It will be understood that a variety of different characteristicsof an α-amylase variant, including specific activity, substratespecificity, K_(m) (the so-called “Michaelis constant” in theMichaelis-Menten equation), V_(max) [the maximum rate (plateau value) ofconversion of a given substrate determined on the basis of theMichaelis-Menten equation], pI, pH optimum, temperature optimum,thermoactivation, stability towards oxidants or surfactants (e.g. indetergents), etc., taken alone or in combination, can contribute toimproved performance. The skilled person will be aware that theperformance of the variant cannot, alone, be predicted on the basis ofthe above characteristics, but would have to be accompanied by washingand/or dishwashing performance tests.

[0036] In further aspects the invention relates to a DNA constructcomprising a DNA sequence encoding an α-amylase variant of theinvention, a recombinant expression vector carrying the DNA construct, acell which is transformed with the DNA construct or the vector, as wellas a method of producing an α-amylase variant by culturing such a cellunder conditions conducive to the production of the α-amylase variant,after which the α-amylase variant is recovered from the culture.

[0037] In a further aspect the invention relates to a method ofpreparing a variant of a parent α-amylase which by virtue of itsimproved properties as described above exhibits improved washing and/ordishwashing performance as compared to the parent α-amylase. This methodcomprises

[0038] a) constructing a population of cells containing genes encodingvariants of said parent α-amylase,

[0039] b) screening the population of cells for α-amylase activity underconditions simulating at least one washing and/or dishwashing condition,

[0040] c) isolating a cell from the population containing a geneencoding a variant of said parent α-amylase which has improved activityas compared with the parent α-amylase under the conditions selected instep b),

[0041] d) culturing the cell isolated in step c) under suitableconditions in an appropriate culture medium, and

[0042] e) recovering the α-amylase variant from the culture obtained instep d).

[0043] The invention also relates to a variant (which is a variantaccording the invention) prepared by the latter method.

[0044] In the present context, the term “simulating at least one washingand/or dishwashing condition” is intended to indicate a simulation of,e.g., the temperature or pH prevailing during washing or dishwashing, orof the chemical composition of a detergent composition to be used in thewashing or dishwashing treatment. The term “chemical composition” isintended to include one, or a combination of two or more, constituentsof the detergent composition in question. The constituents of a numberof different detergent compositions are listed further below.

[0045] The “population of cells” referred to in step a) may suitably beconstructed by cloning a DNA sequence encoding a parent α-amylase andsubjecting the DNA to site-directed or random mutagenesis as describedherein.

[0046] In the present context the term “variant” is used interchangeablywith the term “mutant”. The term “variant” is intended to include hybridα-amylases, i.e. α-amylases comprising parts of at least two differentα-amylolytic enzymes. Thus, such a hybrid may be constructed, e.g.,from: one or more parts each deriving from a variant as already definedabove; or one or more parts each deriving from a variant as alreadydefined above, and one or more parts each deriving from an unmodifiedparent α-amylase. In this connection, the invention also relates to amethod of producing such a hybrid α-amylase having improved washingand/or dishwashing performance as compared to any of its constituentenzymes (i.e. as compared to any of the enzymes which contribute a partto the hybrid), which method comprises:

[0047] a) recombining in vivo or in vitro the N-terminal coding regionof an α-amylase gene or corresponding cDNA of one of the constituentα-amylases with the C-terminal coding region of an α-amylase gene orcorresponding cDNA of another constituent α-amylase to formrecombinants,

[0048] b) selecting recombinants that produce a hybrid α-amylase havingimproved washing and/or dishwashing performance as compared to any ofits constituent α-amylases,

[0049] c) culturing recombinants selected in step b) under suitableconditions in an appropriate culture medium, and

[0050] d) recovering the hybrid α-amylase from the culture obtained instep c).

[0051] In further aspects the invention relates to the use of anα-amylase variant of the invention [including any variant or hybridprepared by one of the above mentioned methods] as a detergent enzyme,in particular for washing or dishwashing, to a detergent additive and adetergent composition comprising the α-amylase variant, and to the useof an α-amylase variant of the invention for textile desizing.

[0052] Random mutagenesis may be used to generate variants according tothe invention, and the invention further relates to a method ofpreparing a variant of a parent α-amylase, which method comprises

[0053] (a) subjecting a DNA sequence encoding the parent α-amylase torandom mutagenesis,

[0054] (b) expressing the mutated DNA sequence obtained in step (a) in ahost cell, and

[0055] (c) screening for host cells expressing a mutated amylolyticenzyme which has improved properties as described above (e.g. propertiessuch as decreased calcium dependency, increased oxidation stability,increased thermostability, and/or improved activity at relatively highpH) as compared to the parent α-amylase.

DETAILED DISCLOSURE OF THE INVENTION

[0056] Nomenclature

[0057] In the present description and claims, the conventionalone-letter codes for nucleotides and the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, α-amylase variants of the invention are described by use ofthe following nomenclature:

[0058] Original amino acid(s):position(s):substituted amino acid(s)

[0059] According to this nomenclature, and by way of example, thesubstitution of alanine for asparagine in position 30 is shown as:

[0060] Ala 30 Asn or A30N

[0061] a deletion of alanine in the same position is shown as:

[0062] Ala 30* or A30*

[0063] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0064] Ala 30 AlaLys or A30AK

[0065] A deletion of a consecutive stretch of amino acid residues,exemplified by amino acid residues 30-33, is indicated as (30-33)*.

[0066] Where a specific α-amylase contains a “deletion” (i.e. lacks anamino acid residue) in comparison with other α-amylases and an insertionis made in such a position, this is indicated as:

[0067] *36 Asp or *36D

[0068] for insertion of an aspartic acid in position 36.

[0069] Multiple mutations are separated by plus signs, i.e.:

[0070] Ala 30 Asp+Glu 34 Ser or A30N+E34S

[0071] representing mutations in positions 30 and 34 (in which alanineand glutamic acid replace, i.e. are substituted for, asparagine andserine, respectively).

[0072] When one or more alternative amino acid residues may be insertedin a given position this is indicated as:

[0073] A30N,E or

[0074] A30N or A30E

[0075] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any other amino acid residue may be substitutedfor the amino acid residue present in that position (i.e. any amino acidresidue—other than that normally present in the position inquestion—chosen among A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T,W, Y and V). Thus, for instance, when a modification (replacement) of amethionine in position 202 is mentioned, but not specified, it is to beunderstood that any of the other amino acids may be substituted for themethionine, i.e. any other amino acid chosen among A, R, N, D, C, Q, E,G, H, I, L, K, F, P, S, T, W, Y and V.

[0076] The Parent α-Amylase

[0077] As already indicated, an α-amylase variant of the invention isvery suitably prepared on the basis of a parent α-amylase having one ofthe amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No.3 and SEQ ID No. 7, respectively (vide infra).

[0078] The parent α-amylases having the amino acid sequences shown inSEQ ID No. 1 and SEQ ID No. 2, respectively, are obtainable fromalkalophilic Bacillus strains (strain NCIB 12512 and strain NCIB 12513,respectively), both of which are described in detail in EP 0 277 216 B1.The preparation, purification and sequencing of these two parentα-amylases is described in WO 95/26397 [see the Experimental sectionherein (vide infra)].

[0079] The parent α-amylase having the amino acid sequence shown in SEQID No. 3 is obtainable from Bacillus stearothermophilus and is describedin, inter alia, J. Bacteriol. 166 (1986) pp. 635-643.

[0080] The parent α-amylase having the amino acid sequence shown in SEQID No. 7 (which is the same sequence as that numbered 4 in FIG. 1) isobtainable from a “Bacillus sp. #707” and is described by Tsukamoto etal. in Biochem. Biophys. Res. Commun. 151 (1988) pp. 25-31.

[0081] Apart from variants of the above-mentioned parent α-amylaseshaving the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQID No. 3 and SEQ ID No. 7, respectively, other interesting variantsaccording to the invention include variants of parent α-amylases whichhave amino acid sequences exhibiting a high degree of homology, such asat least 70% homology, preferably (as already indicated) at least 80%homology, desirably at least 85% homology, and more preferably at least90% homology, e.g. 095% homology, with at least one of the latter fouramino acid sequences.

[0082] As also already indicated above, further criteria for identifyinga suitable parent α-amylase are a) that the α-amylase displays animmunological cross-reaction with an antibody raised against anα-amylase having one of the amino acid sequences shown in SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively, and/or b)that the α-amylase is encoded by a DNA sequence which hybridizes withthe same probe as a DNA sequence encoding an α-amylase having one of theamino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3and SEQ ID No. 7, respectively.

[0083] As already mentioned, with regard to determination of the degreeof homology of polypeptides (such as enzymes), amino acid sequencecomparisons can be performed using known algorithms, such as the onedescribed by Lipman and Pearson (1985).

[0084] Assays for immunological cross-reactivity may be carried outusing an antibody raised against, or reactive with, at least one epitopeof the α-amylase having the amino acid sequence shown in SEQ ID No. 1,or of the α-amylase having the amino acid sequence shown in SEQ ID No.2, or of the α-amylase having the amino acid sequence shown in SEQ IDNo. 3, or of the α-amylase having the amino acid sequence shown in SEQID No. 7.

[0085] The antibody, which may either be monoclonal or polyclonal, maybe produced by methods known in the art, e.g. as described by Hudson etal. (1989). Examples of suitable assay techniques well known in the artinclude Western Blotting and Radial Immunodiffusion Assay, e.g. asdescribed by Hudson et al. (1989).

[0086] The oligonucleotide probe for use in the identification ofsuitable parent α-amylases on the basis of probe hybridization[criterion b) above] may, by way of example, suitably be prepared on thebasis of the full or partial amino acid sequence of an α-amylase havingone of the sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3and SEQ ID No. 7, respectively, or on the basis of the full or partialnucleotide sequence corresponding thereto.

[0087] Suitable conditions for testing hybridization involve presoakingin 5×SSC and prehybridizing for 1 h at ˜40□C in a solution of 20%formamide, 5× Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and50 μg of denatured sonicated calf thymus DNA, followed by hybridizationin the same solution supplemented with 100 μM ATP for 18 h at ˜40□C, orusing other methods described by, e.g., Sambrook et al. (1989).

[0088] Influence of Mutations on Particular Properties

[0089] From the results obtained by the present inventors it appearsthat changes in a particular property, e.g. thermal stability oroxidation stability, exhibited by a variant relative to the parentα-amylase in question can to a considerable extent be correlated withthe type of, and positioning of, mutation(s) (amino acid substitutions,deletions or insertions) in the variant. It is to be understood,however, that the observation that a particular mutation or pattern ofmutations leads to changes in a given property in no way excludes thepossibility that the mutation(s) in question can also influence otherproperties.

[0090] Oxidation stability: With respect to increasing the oxidationstability of an α-amylase variant relative to its parent α-amylase, itappears to be particularly desirable that at least one, and preferablymultiple, oxidizable amino acid residue(s) of the parent has/have beendeleted or replaced (i.e. substituted by) a different amino acid residuewhich is less susceptible to oxidation than the original oxidizableamino acid residue.

[0091] Particularly relevant oxidizable amino acid residues in thisconnection are cysteine, methionine, tryptophan and tyrosine. Thus, forexample, in the case of parent α-amylases containing cysteine it isanticipated that deletion of cysteine residues, or substitution thereofby less oxidizable amino acid residues, will be of importance inobtaining variants with improved oxidation stability relative to theparent α-amylase.

[0092] In the case of the above-mentioned parent α-amylases having theamino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.7, respectively, all of which contain no cysteine residues but have asignificant methionine content, the deletion or substitution ofmethionine residues is particularly relevant with respect to achievingimproved oxidation stability of the resulting variants. Thus, deletionor substitution [e.g. by threonine (T), or by one of the other aminoacids listed above] of one or more of the methionine residues inpositions M9, M10, M105, M202, M208, M261, M309, M382, M430 and M440 ofthe amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ IDNo. 7, and/or in position M323 of the amino acid sequence shown in SEQID No. 2 (or deletion or substitution of methionine residues inequivalent positions in the sequence of another α-amylase meeting one ofthe other criteria for a parent α-amylase mentioned above) appear to beparticularly effective with respect to increasing the oxidationstability.

[0093] In the case of the parent α-amylase having the amino acidsequence shown in SEQ ID No. 3, relevant amino acid residues which maybe deleted or substituted with a view to improving the oxidationstability include the single cysteine residue (C363) and—by analogy withthe sequences shown in SEQ ID No. 1 and SEQ ID No. 3—the methionineresidues located in positions M8, M9, M96, M200, M206, M284, M307, M311,M316 and M438.

[0094] In this connection, the term “equivalent position” denotes aposition which, on the basis of an alignment of the amino acid sequenceof the parent α-amylase in question with the “reference” α-amylase aminoacid sequence in question (for example the sequence shown in SEQ IDNo. 1) so as to achieve juxtapositioning of amino acid residues/regionswhich are common to both, corresponds most closely to (e.g. is occupiedby the same amino acid residue as) a particular position in thereference sequence in question.

[0095] Particularly interesting mutations in connection withmodification (improvement) of the oxidation stability of the α-amylaseshaving the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 andSEQ ID No. 7, respectively, are one or more of the following methioninesubstitutions (or equivalents thereof in the amino acid sequences ofother α-amylases meeting the requirements of a parent α-amylase in thecontext of the invention): M202A, R, N, D, Q, E, G, H, I, L, K, F, P, S,T, W, Y, V.

[0096] Further relevant methionine substitutions in the amino acidsequence shown in SEQ ID No. 2 are: M323A, R, N, D, Q, E, G, H, I, L, K,F, P, S, T, W, Y, V.

[0097] Particularly interesting mutations in connection withmodification (improvement) of the oxidation stability of the α-amylasehaving the amino acid sequence shown in SEQ ID No. 3 are one or more ofthe following methionine substitutions:

[0098] M200A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V;

[0099] M311A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V; and

[0100] M316A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V.

[0101] Thermal stability: With respect to increasing the thermalstability of an α-amylase variant relative to its parent α-amylase, itappears to be particularly desirable to delete at least one, andpreferably two or even three, of the following amino acid residues inthe amino acid sequence shown in SEQ ID No. 1 (or their equivalents):F180, R181, G182, T183, G184 and K185. The corresponding, particularlyrelevant (and equivalent) amino acid residues in the amino acidsequences shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7,respectively, are: F180, R181, G182, D183, G184 and K185 (SEQ ID No. 2);F178, R179, G180, I181, G182 and K183 (SEQ ID No. 3); and F180, R181,G182, H183, G184 and K185 (SEQ ID No. 7).

[0102] Particularly interesting pairwise deletions of this type are asfollows:

[0103] R181*+G182*; and T183*+G184* (SEQ ID No. 1);

[0104] R181*+G182*; and D183*+G184* (SEQ ID No. 2);

[0105] R179*+G180*; and 1181*+G182* (SEQ ID No. 3); and

[0106] R181*+G182*; and H183*+G184* (SEQ ID No. 7)

[0107] (or equivalents of these pairwise deletions in another α-amylasemeeting the requirements of a parent α-amylase in the context of thepresent invention).

[0108] Other mutations which appear to be of importance in connectionwith thermal stability are substitutions of one or more of the aminoacid residues from P260 to 1275 in the sequence shown in SEQ ID No. 1(or equivalents thereof in another parent α-amylase in the context ofthe invention), such as substitution of the lysine residue in position269.

[0109] Examples of specific mutations which appear to be of importancein connection with the thermal stability of an α-amylase variantrelative to the parent α-amylase in question are one or more of thefollowing substitutions in the amino acid sequence shown in SEQ ID No. 1(or equivalents thereof in another parent α-amylase in the context ofthe invention): K269R; P260E; R124P; M105F,I,L,V; M208F,W,Y; L2171;V206I,L,F.

[0110] For the parent α-amylase having the amino acid sequence shown inSEQ ID No. 2, important further (equivalent) mutations are,correspondingly, one or more of the substitutions: M105F,I,L,V;M208F,W,Y; L2171; V206I,L,F; and K269R.

[0111] For the parent α-amylase having the amino acid sequence shown inSEQ ID No. 3, important further (equivalent) mutations are,correspondingly, one or both of the substitutions: M206F,W,Y; and L215I.

[0112] For the parent α-amylase having the amino acid sequence shown inSEQ ID No. 7, important further (equivalent) mutations are,correspondingly, one or more of the substitutions: M105F,I,L,V;M208F,W,Y; L2171; and K269R.

[0113] Still further examples of mutations which appear to be ofimportance, inter alia, in achieving improved thermal stability of anα-amylase variant relative to the parent α-amylase in question are oneor more of the following substitutions in the amino acid sequences shownin SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7 (or equivalents thereofin another parent α-amylase in the context of the invention):A354C+V479C; L351C+M430C; N457D,E+K385R; L355D,E+M430R,K;L355D,E+I411R,K; and N457D,E.

[0114] Ca²⁺ dependency: With respect to achieving decreased Ca²⁺dependency of an α-amylase variant relative to its parent α-amylase[i.e. with respect to obtaining a variant which exhibits satisfactoryamylolytic activity in the presence of a lower concentration of calciumion in the extraneous medium than is necessary for the parent enzyme,and which, for example, therefore is less sensitive than the parent tocalcium ion-depleting conditions such as those obtaining in mediacontaining calcium-complexing agents (such as certain detergentbuilders)], it appears to be particularly desirable to incorporate oneor more of the following substitutions in the amino acid sequences shownin SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7 (or an equivalentsubstitution in another parent α-amylase in the context of theinvention): Y243F, K108R, K179R, K239R, K242R, K269R, D163N, D188N,D192N, D199N, D205N, D207N, D209N, E190Q, E194Q and N₁O₆D.

[0115] In the case of the amino acid sequence shown in SEQ ID No. 3,particularly desirable substitutions appear, correspondingly(equivalently), to be one or more of the following: K107R, K177R, K237R,K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N, E188Q and E192Q.

[0116] As well as the above-mentioned replacements of D residues with Nresidues, or of E residues with Q residues, other relevant substitutionsin the context of reducing Ca²⁺ dependency are replacement of the Dand/or E residues in question with any other amino acid residue.

[0117] Further substitutions which appear to be of importance in thecontext of achieving reduced Ca²⁺ dependency are pairwise substitutionsof the amino acid residues present at: positions 113 and 151, andpositions 351 and 430, in the amino acid sequences shown in SEQ ID No.1, SEQ ID No. 2 and SEQ ID No. 7; and at: positions 112 and 150, andpositions 349 and 428, in the amino acid sequence shown in SEQ ID No. 3(or equivalent pairwise substitutions in another parent α-amylase in thecontext of the invention), i.e. pairwise substitutions of the followingamino acid residues:

[0118] G113+N151 (in relation to SEQ ID No. 1); A113+T151 (in relationto SEQ ID No. 2 and SEQ ID No. 7); and Gi 12+T150 (in relation to SEQ IDNo. 3); and

[0119] L351+M430 (in relation to SEQ ID No. 1, SEQ ID No. 2 and SEQ IDNo. 7); and L349+1428 (in relation to SEQ ID No. 3).

[0120] Particularly interesting pairwise substitutions of this type withrespect to achieving decreased Ca²⁺ dependency are the following:

[0121] G113T+N151I (in relation to SEQ ID No. 1); A113T+T151I (inrelation to SEQ ID No. 2 and SEQ ID No. 7); and G112T+T150I (in relationto SEQ ID No. 3); and L351C+M430C (in relation to SEQ ID No. 1, SEQ IDNo. 2 and SEQ ID No. 7); and L349C+1428C (in relation to SEQ ID No. 3).

[0122] In connection with substitutions of relevance for Ca²⁺dependency, some other substitutions which appear to be of importance instabilizing the enzyme conformation, and which it is contemplated mayachieve this by, e.g., enhancing the strength of binding or retention ofcalcium ion at or within a calcium binding site within the α-amylolyticenzyme, are one or more of the following substitutions in the amino acidsequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 7 (or anequivalent substitution in another parent α-amylase in the context ofthe invention): G304W,F,Y,R,I,L,V,Q,N; G305A,S,N,D,Q,E,R,K; and H408Q,E.

[0123] Corresponding (equivalent) substitutions in the amino acidsequence shown in SEQ ID No. 3 are: G302W,F,Y,R,I,L,V,Q,N; andG303A,S,N,D,Q,E,R,K.

[0124] Further mutations which appear to be of importance in the contextof achieving reduced Ca²⁺ dependency are pairwise deletions of aminoacids (i.e. deletion of two amino acids) at positions selected amongR181, G182, T183 and G184 in the amino acid sequence shown in SEQ ID No.1 (or equivalent positions in the amino acid sequence of anotherα-amylase meeting the requirements of a parent α-amylase in the contextof the invention). Such pairwise deletions are thus the following:

[0125] R181*+G182*; T183*+G184*; R181*+T183*; G182*+T183*; G182*+G184*;and R181*+G184* (SEQ ID No. 1);

[0126] R181*+G182*; D183*+G184*; R181*+D183*; G182*+D183*; G182*+G184*;and R181*+G184* (SEQ ID No. 2);

[0127] R179*+G180*; 1181*+G182*; R179*+I181*; G180*+I181*; G180*+G182*;and R179*+G182* (SEQ ID No. 3); and

[0128] R181*+G182*; H183*+G184*; R181*+H183*; G182*+H183*; G182*+G184*;and R181*+G184* (SEQ ID No. 7);

[0129] (or equivalents of these pairwise deletions in another α-amylasemeeting the requirements of a parent α-amylase in the context of thepresent invention).

[0130] Isoelectric point (pI): Preliminary results indicate that thewashing performance, e.g. the laundry washing performance, of anα-amylase is optimal when the pH of the washing liquor (washing medium)is close to the pI value for the α-amylase in question. It will thus bedesirable, where appropriate, to produce an α-amylase variant having anisoelectric point (pI value) which is better matched to the pH of amedium (such as a washing medium) in which the enzyme is to be employedthan the isoelectric point of the parent α-amylase in question.

[0131] With respect to decreasing the isoelectric point, preferredmutations in the amino acid sequence shown in SEQ ID No. 1 include oneor more of the following substitutions: Q86E, R124P, S154D, T183D,V222E, P260E, R310A, Q346E, Q391E, N437E, K444Q and R452H. Appropriatecombinations of these substitutions in the context of decreasing theisoelectric point include: Q391E+K444Q; and Q391E+K444Q+S154D.

[0132] Correspondingly, preferred mutations in the amino acid sequenceshown in SEQ ID No. 3 with respect to decreasing the isoelectric pointinclude one or more of the substitutions: L85E, S153D, 1181D, K220E,P258E, R308A, P344E, Q358E and S435E.

[0133] With respect to increasing the isoelectric point, preferredmutations in the amino acid sequence shown in SEQ ID No. 2 include oneor more of the following substitutions: E86Q,L; D154S; D183T,I; E222V,K;E260P; A310R; E346Q,P; E437N,S; and H452R.

[0134] In the Experimental section below, the construction of a numberof variants according to the invention is described.

[0135] α-Amylase variants of the invention will, apart from having oneor more improved properties as discussed above, preferably be such thatthey have a higher starch hydrolysis velocity at low substrateconcentrations than the parent α-amylase. Alternatively, an α-amylasevariant of the invention will preferably be one which has a higherV_(max) and/or a lower K_(m) than the parent α-amylase when tested underthe same conditions. In the case of a hybrid α-amylase, the “parentα-amylase” to be used for the comparison should be the one of theconstituent enzymes having the best performance.

[0136] V_(max) and K_(m) (parameters of the Michaelis-Menten equation)may be determined by well-known procedures.

[0137] Methods of Preparing α-Amylase Variants

[0138] Several methods for introducing mutations into genes are known inthe art. After a brief discussion of the cloning of α-amylase-encodingDNA sequences, methods for generating mutations at specific sites withinthe α-amylase-encoding sequence will be discussed.

[0139] Cloning a DNA Sequence Encoding an α-Amylase

[0140] The DNA sequence encoding a parent α-amylase may be isolated fromany cell or microorganism producing the α-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the α-amylase to be studied. Then, ifthe amino acid sequence of the α-amylase is known, homologous, labelledoligonucleotide probes may be synthesized and used to identifyα-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to a known α-amylase gene could be usedas a probe to identify α-amylase-encoding clones, using hybridizationand washing conditions of lower stringency.

[0141] Yet another method for identifying α-amylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming α-amylase-negative bacteria withthe resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for α-amylase, therebyallowing clones expressing the α-amylase to be identified.

[0142] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g. thephosphoamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[0143] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genom ic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al. (1988).

[0144] Site-Directed Mutagenesis

[0145] Once an α-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging the c-amylase-encodingsequence, is created in a vector carrying the α-amylase gene. Then thesynthetic nucleotide, bearing the desired mutation, is annealed to ahomologous portion of the single-stranded DNA. The remaining gap is thenfilled in with DNA polymerase I (Klenow fragment) and the construct isligated using T4 ligase. A specific example of this method is describedin Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette. However, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

[0146] Another method of introducing mutations into α-amylase-encodingDNA sequences is described in Nelson and Long (1989). It involves the3-step generation of a PCR fragment containing the desired mutationintroduced by using a chemically synthesized DNA strand as one of theprimers in the PCR reactions. From the PCR-generated fragment, a DNAfragment carrying the mutation may be isolated by cleavage withrestriction endonucleases and reinserted into an expression plasmid.

[0147] Random Mutagenesis

[0148] Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

[0149] For region-specific random mutagenesis with a view to improvingthe thermal stability, the following codon positions, in particular, mayappropriately be targeted (using one-letter amino acid abbreviations andthe numbering of the amino acid residues in the sequence in question):

[0150] In the Amino Acid Sequence Shown in SEQ ID No. 1:

[0151] 120-140=VEVNRSNRNQETSGEYAIEAW

[0152] 178-187=YKFRGTGKAW

[0153] 264-277=VAEFWKNDLGAIEN

[0154] In the Amino Acid Sequence Shown in SEQ ID No. 2:

[0155] 120-140=VEVNPNNRNQEISGDYTIEAW

[0156] 178-187=YKFRGDGKAW

[0157] 264-277=VAEFWKNDLGALEN

[0158] In the Amino Acid Sequence Shown in SEQ ID No. 3:

[0159] 119-139=VEVNPSDRNQEISGTYQIQAW

[0160] 176-185=YKFRGIGKAW

[0161] 262-275=VGEYWSYDINKLHN

[0162] In the Amino Acid Sequence Shown in SEQ ID No. 7:

[0163] 120-140=VEVNPNNRNQEVTGEYTIEAW

[0164] 178-187=YKFRGHGKAW

[0165] 264-277=VAEFWKNDLGAIEN

[0166] With a view to achieving reduced Ca²⁺ dependency, the followingcodon positions, in particular, may appropriately be targeted:

[0167] In the Amino Acid Sequence Shown in SEQ ID No. 1:

[0168] 178-209=YKFRGTGKAWDWEVDTENGNYDYLMYADVDMD

[0169] 237-246=AVKHIKYSFT

[0170] In the Amino Acid Sequence Shown in SEQ ID No. 2:

[0171] 178-209=YKFRGDGKAWDWEVDSENGNYDYLMYADVDMD

[0172] 237-246=AVKHIKYSFT

[0173] In the Amino Acid Sequence Shown in SEQ ID No. 7:

[0174] 178-209=YKFRGHGKAWDWEVDTENGNYDYLMYADIDMD

[0175] 237-246=AVKHIKYSFT

[0176] With a view to achieving improved binding of a substrate (i.e.improved binding of a carbohydrate species—such as amylose oramylopectin—which is a substrate for α-amylolytic enzymes) by anα-amylase variant, modified (e.g. higher) substrate specificity and/ormodified (e.g. higher) specificity with respect to cleavage (hydrolysis)of substrate, it appears that the following codon positions for theamino acid sequence shown in SEQ ID No. 1 (or equivalent codon positionsfor another parent α-amylase in the context of the invention) mayparticularly appropriately be targeted:

[0177] In the Amino Acid Sequence Shown in SEQ ID No. 1:

[0178] 15-20=WYLPND

[0179] 52-58=SQNDVGY

[0180] 72-78=KGTVRTK

[0181] 104-111=VMNHKGGA

[0182] 165-174=TDWDQSRQLQ

[0183] 194-204=ENGNYDYLMYA

[0184] 234-240=RIDAVKH

[0185] 332-340=HDSQPGEAL

[0186] The random mutagenesis of a DNA sequence encoding a parentα-amylase to be performed in accordance with step a) of theabove-described method of the invention may conveniently be performed byuse of any method known in the art.

[0187] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents.

[0188] The mutagenizing agent may, e.g., be one which inducestransitions, transversions, inversions, scrambling, deletions, and/orinsertions.

[0189] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues.

[0190] When such agents are used, the mutagenesis is typically performedby incubating the DNA sequence encoding the parent enzyme to bemutagenized in the presence of the mutagenizing agent of choice undersuitable conditions for the mutagenesis to take place, and selecting formutated DNA having the desired properties.

[0191] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding theamylolytic enzyme by any published technique, using e.g. PCR, LCR or anyDNA polymerase and ligase.

[0192] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent α-amylase enzyme issubjected to PCR under conditions that increase the misincorporation ofnucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp.11-15).

[0193] A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding theamylolytic enzyme by e.g. transforming a plasmid containing the parentenzyme into the mutator strain, growing the mutator strain with theplasmid and isolating the mutated plasmid from the mutator strain. Themutated plasmid may subsequently be transformed into the expressionorganism.

[0194] The DNA sequence to be mutagenized may conveniently be present ina genomic or cDNA library prepared from an organism expressing theparent amylolytic enzyme. Alternatively, the DNA sequence may be presenton a suitable vector such as a plasmid or a bacteriophage, which as suchmay be incubated with or otherwise exposed to the mutagenizing agent.The DNA to be mutagenized may also be present in a host cell either bybeing integrated in the genome of said cell or by being present on avector harbored in the cell. Finally, the DNA to be mutagenized may bein isolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0195] In some cases it may be convenient to amplify the mutated DNAsequence prior to the expression step (b) or the screening step (c)being performed. Such amplification may be performed in accordance withmethods known in the art, the presently preferred method beingPCR-generated amplification using oligonucleotide primers prepared onthe basis of the DNA or amino acid sequence of the parent enzyme.

[0196] Subsequent to the incubation with or exposure to the mutagenizingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which was carried the DNA sequence encoding the parentenzyme during the mutagenesis treatment. Examples of suitable host cellsare the following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram negative bacteria such as E. coli.

[0197] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

[0198] Localized random mutagenesis: the random mutagenesis mayadvantageously be localized to a part of the parent α-amylase inquestion. This may, e.g., be advantageous when certain regions of theenzyme have been identified to be of particular importance for a givenproperty of the enzyme, and when modified are expected to result in avariant having improved properties. Such regions may normally beidentified when the tertiary structure of the parent enzyme has beenelucidated and related to the function of the enzyme.

[0199] The localized random mutagenesis is conveniently performed by useof PCR-generated mutagenesis techniques as described above or any othersuitable technique known in the art.

[0200] Alternatively, the DNA sequence encoding the part of the DNAsequence to be modified may be isolated, e.g. by being inserted into asuitable vector, and said part may subsequently be subjected tomutagenesis by use of any of the mutagenesis methods discussed above.

[0201] With respect to the screening step in the above-mentioned methodof the invention, this may conveniently performed by use of a filterassay based on the following principle:

[0202] A microorganism capable of expressing the mutated amylolyticenzyme of interest is incubated on a suitable medium and under suitableconditions for the enzyme to be secreted, the medium being provided witha double filter comprising a first protein-binding filter and on top ofthat a second filter exhibiting a low protein binding capability. Themicroorganism is located on the second filter. Subsequent to theincubation, the first filter comprising enzymes secreted from themicroorganisms is separated from the second filter comprising themicroorganisms. The first filter is subjected to screening for thedesired enzymatic activity and the corresponding microbial coloniespresent on the second filter are identified.

[0203] The filter used for binding the enzymatic activity may be anyprotein binding filter e.g. nylon or nitrocellulose. The top filtercarrying the colonies of the expression organism may be any filter thathas no or low affinity for binding proteins e.g. cellulose acetate orDurapore™. The filter may be pretreated with any of the conditions to beused for screening or may be treated during the detection of enzymaticactivity.

[0204] The enzymatic activity may be detected by a dye, flourescence,precipitation, pH indicator, IR-absorbance or any other known techniquefor detection of enzymatic activity.

[0205] The detecting compound may be immobilized by any immobilizingagent e.g. agarose, agar, gelatine, polyacrylamide, starch, filterpaper, cloth; or any combination of immobilizing agents.

[0206] α-Amylase activity is detected by Cibacron Red labelledamylopectin, which is immobilized on agarose. For screening for variantswith increased thermal and high-pH stability, the filter with boundα-amylase variants is incubated in a buffer at pH 10.5 and 60□ or 65□Cfor a specified time, rinsed briefly in deionized water and placed onthe amylopectin-agarose matrix for activity detection. Residual activityis seen as lysis of Cibacron Red by amylopectin degradation. Theconditions are chosen to be such that activity due to the α-amylasehaving the amino acid sequence shown in SEQ ID No. 1 can barely bedetected. Stabilized variants show, under the same conditions, increasedcolor intensity due to increased liberation of Cibacron Red.

[0207] For screening for variants with an activity optimum at a lowertemperature and/or over a broader temperature range, the filter withbound variants is placed directly on the amylopectin-Cibacron Redsubstrate plate and incubated at the desired temperature (e.g. 40C, 10□Cor 30□C) for a specified time. After this time activity due to theα-amylase having the amino acid sequence shown in SEQ ID No. 1 canbarely be detected, whereas variants with optimum activity at a lowertemperature will show increase amylopectin lysis. Prior to incubationonto the amylopectin matrix, incubation in all kinds of desiredmedia—e.g. solutions containing Ca²⁺, detergents, EDTA or other relevantadditives—can be carried out in order to screen for changed dependencyor for reaction of the variants in question with such additives.

[0208] Methods of Preparing Hybrid α-Amylases

[0209] As an alternative to site-specific mutagenesis, α-amylasevariants which are hybrids of at least two constituent α-amylases may beprepared by combining the relevant parts of the respective genes inquestion.

[0210] Naturally occurring enzymes may be genetically modified by randomor site directed mutagenesis as described above. Alternatively, part ofone enzyme may be replaced by a part of another to obtain a chimericenzyme. This replacement can be achieved either by conventional in vitrogene splicing techniques or by in vivo recombination or by combinationsof both techniques. When using conventional in vitro gene splicingtechniques, a desired portion of the α-amylase gene coding sequence maybe deleted using appropriate site-specific restriction enzymes; thedeleted portion of the coding sequence may then be replaced by theinsertion of a desired portion of a different α-amylase coding sequenceso that a chimeric nucleotide sequence encoding a new α-amylase isproduced. Alternatively, α-amylase genes may be fused, e.g. by use ofthe PCR overlay extension method described by Higuchi et al. 1988.

[0211] The in vivo recombination techniques depend on the fact thatdifferent DNA segments with highly homologous regions (identity of DNAsequence) may recombine, i.e. break and exchange DNA, and establish newbonds in the homologous regions. Accordingly, when the coding sequencesfor two different but homologous amylase enzymes are used to transform ahost cell, recombination of homologous sequences in vivo will result inthe production of chimeric gene sequences. Translation of these codingsequences by the host cell will result in production of a chimericamylase gene product. Specific in vivo recombination techniques aredescribed in U.S. Pat. No. 5,093,257 and EP 252 666.

[0212] Alternatively, the hybrid enzyme may be synthesized by standardchemical methods known in the art. For example, see Hunkapiller et al.(1984). Accordingly, peptides having the appropriate amino acidsequences may be synthesized in whole or in part and joined to formhybrid enzymes (variants) of the invention.

[0213] Expression of α-Amylase Variants

[0214] According to the invention, a mutated α-amylase-encoding DNAsequence produced by methods described above, or by any alternativemethods known in the art, can be expressed, in enzyme form, using anexpression vector which typically includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene or various activator genes.

[0215] The recombinant expression vector carrying the DNA sequenceencoding an α-amylase variant of the invention may be any vector whichmay conveniently be subjected to recombinant DNA procedures, and thechoice of vector will often depend on the host cell into which it is tobe introduced. Thus, the vector may be an autonomously replicatingvector, i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[0216] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an α-amylase variant of theinvention, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus Amyloliquefaciensα-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylBgenes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger glucoamylase,Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase or A. nidulans acetamidase.

[0217] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding the α-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

[0218] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

[0219] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

[0220] While intracellular expression may be advantageous in somerespects, e.g. when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular.

[0221] Procedures suitable for constructing vectors of the inventionencoding an α-amylase variant, and containing the promoter, terminatorand other elements, respectively, are well known to persons skilled inthe art [cf., for instance, Sambrook et al. (1989)].

[0222] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of anα-amylase variant of the invention. The cell may be transformed with theDNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0223] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

[0224] Examples of suitable bacteria are gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

[0225] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[0226] In a yet further aspect, the present invention relates to amethod of producing an α-amylase variant of the invention, which methodcomprises cultivating a host cell as described above under conditionsconducive to the production of the variant and recovering the variantfrom the cells and/or culture medium.

[0227] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the α-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. as described in catalogues of the American TypeCulture Collection).

[0228] The α-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

INDUSTRIAL APPLICATIONS

[0229] Owing to their activity at alkaline pH values, α-amylase variantsof the invention are well suited for use in a variety of industrialprocesses. In particular, they find potential applications as acomponent in washing, dishwashing and hard surface cleaning detergentcompositions (vide infra), but may also be useful in the production ofsweeteners and ethanol from starch. Conditions for conventionalstarch-converting processes and liquefaction and/or saccharificationprocesses are described in, for instance, U.S. Pat. No. 3,912,590, EP252,730 and EP 63,909.

[0230] Some areas of application of α-amylase variants of the inventionare outlined below.

[0231] Paper-related applications: α-Amylase variants of the inventionpossess properties of value in the production of lignocellulosicmaterials, such as pulp, paper and cardboard, from starch-reinforcedwaste paper and waste cardboard, especially where repulping occurs at apH above 7, and where amylases can facilitate the disintegration of thewaste material through degradation of the reinforcing starch.

[0232] α-Amylase variants of the invention are well suited for use inthe deinking/recycling processes of making paper out of starch-coated orstarch-containing waste printed paper. It is usually desirable to removethe printing ink in order to produce new paper of high brightness;examples of how the variants of the invention may be used in this wayare described in PCT/DK94/00437.

[0233] α-Amylase variants of the invention may also be very useful inmodifying starch where enzymatically modified starch is used inpapermaking together with alkaline fillers such as calcium carbonate,kaolin and clays. With alkaline α-amylase variants of the invention itis feasible to modify the starch in the presence of the filler, thusallowing for a simpler, integrated process.

[0234] Textile desizing: α-Amylase variants of the invention are alsowell suited for use in textile desizing. In the textile processingindustry, α-amylases are traditionally used as auxiliaries in thedesizing process to facilitate the removal of starch-containing sizewhich has served as a protective coating on weft yarns during weaving.

[0235] Complete removal of the size coating after weaving is importantto ensure optimum results in subsequent processes in which the fabric isscoured, bleached and dyed. Enzymatic starch degradation is preferredbecause it does not harm the fibers of the textile or fabric.

[0236] In order to reduce processing costs and increase mill throughput,the desizing processing is sometimes combined with the scouring andbleaching steps. In such cases, non-enzymatic auxiliaries such as alkalior oxidation agents are typically used to break down the starch, becausetraditional α-amylases are not very compatible with high pH levels andbleaching agents. The non-enzymatic breakdown of the starch size doeslead to some fibre damage because of the rather aggressive chemicalsused.

[0237] α-Amylase variants of the invention exhibiting improvedstarch-degrading performance at relatively high pH levels and in thepresence of oxidizing (bleaching) agents are thus well suited for use indesizing processes as described above, in particular for replacement ofnon-enzymatic desizing agents currently used. The α-amylase variant maybe used alone, or in combination with a cellulase when desizingcellulose-containing fabric or textile.

[0238] Beer production: α-Amylase variants of the invention are alsobelieved to be very useful in beer-making processes; in such processesthe variants will typically be added during the mashing process.

[0239] Applications in detergent additives and detergent compositionsfor washing or dishwashing: Owing to the improved washing and/ordishwashing performance which will often be a consequence ofimprovements in properties as discussed above, numerous α-amylasevariants (including hybrids) of the invention are particularly wellsuited for incorporation into detergent compositions, e.g. detergentcompositions intended for performance in the pH range of 7-13,particularly the pH range of 8-11. According to the invention, theα-amylase variant may be added as a component of a detergentcomposition. As such, it may be included in the detergent composition inthe form of a detergent additive.

[0240] Thus, a further aspect of the invention relates to a detergentadditive comprising an α-amylase variant according to the invention. Theenzymes may be included in a detergent composition by adding separateadditives containing one or more enzymes, or by adding a combinedadditive comprising all of these enzymes. A detergent additive of theinvention, i.e. a separated additive or a combined additive, can beformulated, e.g., as a granulate, liquid, slurry, etc. Preferred enzymeformulations for detergent additives are granulates (in particularnon-dusting granulates), liquids (in particular stabilized liquids),slurries or protected enzymes (vide infra).

[0241] The detergent composition as well as the detergent additive mayadditionally comprise one or more other enzymes conventionally used indetergents, such as proteases, lipases, amylolytic enzymes, oxidases(including peroxidases), or cellulases.

[0242] It has been found that substantial improvements in washing and/ordishwashing performance may be obtained when α-amylase is combined withanother amylolytic enzyme, such as a pullulanase, an iso-amylase, abetα-amylase, an amyloglucosidase or a CGTase. Examples of commerciallyavailable amylolytic enzymes suitable for the given purpose are AMG□,Novamyl□ and Promozyme□, all of which available from Novo Nordisk A/S,Bagsvaerd, Denmark. Accordingly, a particular embodiment of theinvention relates to a detergent additive comprising an α-amylasevariant of the invention in combination with at least one otheramylolytic enzyme (e.g. chosen amongst those mentioned above).

[0243] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452, and may optionallybe coated by methods known in the art; further details concerningcoatings are given below. When a combination of different detergentenzymes is to be employed, the enzymes may be mixed before or aftergranulation.

[0244] Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Other enzymestabilizers are well known in the art. Protected enzymes may be preparedaccording to the method disclosed in EP 238 216.

[0245] As already indicated, a still further aspect of the inventionrelates to a detergent composition, e.g. for laundry washing, fordishwashing or for hard-surface cleaning, comprising an α-amylasevariant (including hybrid) of the invention, and a surfactant.

[0246] The detergent composition of the invention may be in anyconvenient form, e.g. as powder, granules or liquid. A liquid detergentmay be aqueous, typically containing up to 90% of water and 0-20% oforganic solvent, or non-aqueous, e.g. as described in EP Patent 120,659.

[0247] Detergent Compositions

[0248] When an α-amylase variant of the invention is employed as acomponent of a detergent composition (e.g. a laundry washing detergentcomposition, or a dishwashing detergent composition), it may, forexample, be included in the detergent composition in the form of anon-dusting granulate, a stabilized liquid, or a protected enzyme. Asmentioned above, non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to NovoIndustri A/S) and may optionally be coated by methods known in the art.Examples of waxy coating materials are poly(ethylene oxide) products(polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in GB 1483591.

[0249] Enzymes added in the form of liquid enzyme preparations may, asindicated above, be stabilized by, e.g., the addition of a polyol suchas propylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art.

[0250] Protected enzymes for inclusion in a detergent composition of theinvention may be prepared, as mentioned above, according to the methoddisclosed in EP 238,216.

[0251] The detergent composition of the invention may be in anyconvenient form, e.g. as powder, granules, paste or liquid. A liquiddetergent may be aqueous, typically containing up to 70% water and 0-30%organic solvent, or nonaqueous.

[0252] The detergent composition comprises one or more surfactants, eachof which may be anionic, nonionic, cationic, or amphoteric(zwitterionic). The detergent will usually contain 0-50% of anionicsurfactant such as linear alkylbenzenesulfonate (LAS),alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS),alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS),alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, orsoap. It may also contain 040% of nonionic surfactant such as alcoholethoxylate (AEO or AE), alcohol propoxylate, carboxylated alcoholethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

[0253] The detergent composition may additionally comprise one or moreother enzymes, such as pullulanase, esterase, lipase, cutinase,protease, cellulase, peroxidase, or oxidase, e.g., laccase.

[0254] Normally the detergent contains 1-65% of a detergent builder(although some dishwashing detergents may contain even up to 90% of adetergent builder) or complexing agent such as zeolite, diphosphate,triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g. SKS-6 from Hoechst).

[0255] The detergent builders may be subdivided intophosphorus-containing and non-phosphorous-containing types. Examples ofphosphorus-containing inorganic alkaline detergent builders include thewater-soluble salts, especially alkali metal pyrophosphates,orthophosphates, polyphosphates and phosphonates. Examples ofnon-phosphorus-containing inorganic builders include water-solublealkali metal carbonates, borates and silicates, as well as layereddisilicates and the various types of water-insoluble crystalline oramorphous alumino silicates of which zeolites are the best knownrepresentatives.

[0256] Examples of suitable organic builders include alkali metal,ammonium or substituted ammonium salts of succinates, malonates, fattyacid malonates, fatty acid sulphonates, carboxymethoxy succinates,polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates andpolyacetyl carboxylates.

[0257] The detergent may also be unbuilt, i.e. essentially free ofdetergent builder.

[0258] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC; typically in the form of the sodium salt),poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinylalcohol) (PVA), polycarboxylates such as polyacrylates, polymaleates,maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acidcopolymers.

[0259] The detergent composition may contain bleaching agents of thechlorine/bromine-type or the oxygen-type. The bleaching agents may becoated or encapsulated. Examples of inorganic chlorine/bromine-typebleaches are lithium, sodium or calcium hypochlorite or hypobromite aswell as chlorinated trisodium phosphate. The bleaching system may alsocomprise a H₂O₂ source such as perborate or percarbonate which may becombined with a peracid-forming bleach activator such astetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).

[0260] Examples of organic chlorine/bromine-type bleaches areheterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable. The bleaching system mayalso comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

[0261] In dishwashing detergents the oxygen bleaches are preferred, forexample in the form of an inorganic persalt, preferably with a bleachprecursor or as a peroxy acid compound. Typical examples of suitableperoxy bleach compounds are alkali metal perborates, both tetrahydratesand monohydrates, alkali metal percarbonates, persilicates andperphosphates. Preferred activator materials are TAED or NOBS.

[0262] The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative such as, e.g., an aromatic borateester, and the composition may be formulated as described in, e.g., WO92/19709 and WO 92/19708. The enzymes of the invention may also bestabilized by adding reversible enzyme inhibitors, e.g., of the proteintype (as described in EP 0 544 777 B1) or the boronic acid type.

[0263] The detergent may also contain other conventional detergentingredients such as, e.g., fabric conditioners including clays,deflocculant material, foam boosters/foam depressors (in dishwashingdetergents foam depressors), suds suppressors, anti-corrosion agents,soil-suspending agents, anti-soil-redeposition agents, dyes, dehydratingagents, bactericides, optical brighteners, or perfume.

[0264] The pH (measured in aqueous solution at use concentration) willusually be neutral or alkaline, e.g. in the range of 7-11.

[0265] Particular forms of laundry detergent compositions within thescope of the invention include:

[0266] 1) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Linear alkylbenzenesulfonate(calculated as acid)  7-12% Alcohol ethoxysulfate (e.g. C₁₂₋₁₈ alcohol,1-2 EO) or 1-4% alkyl sulfate (e.g. C₁₆₋₁₈) Alcohol ethoxylate (e.g.C₁₄₋₁₅ alcohol, 7 EO) 5-9% Sodium carbonate (as Na₂CO₃) 14-20% Solublesilicate (as Na₂O, 2SiO₂) 2-6% Zeolite (as NaAlSiO₄) 15-22% Sodiumsulfate (as Na₂SO₄) 0-6% Sodium citrate/citric acid (asC₆H₅Na₃O₇/C₆H₈O₇)  0-15% Sodium perborate (as NaBO₃.H₂O) 11-18% TAED2-6% Carboxymethylcellulose 0-2% Polymers (e.g. maleic/acrylic acidcopolymer, PVP, 0-3% PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1%   Minor ingredients (e.g. suds suppressors, perfume, 0-5%optical brightener, photobleach)

[0267] 2) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Linear alkylbenzenesulfonate(calculated as acid)  6-11% Alcohol ethoxysulfate (e.g. C₁₂₋₁₈ alcohol,1-2 EO or 1-3% alkyl sulfate (e.g. C₁₆₋₁₈) Alcohol ethoxylate (e.g.C₁₄₋₁₅ alcohol, 7 EO) 5-9% Sodium carbonate (as Na₂CO₃) 15-21% Solublesilicate (as Na₂O, 2SiO₂) 1-4% Zeolite (as NaAlSiO₄) 24-34% Sodiumsulfate (as Na₂SO₄)  4-10% Sodium citrate/citric acid (asC₆H₅Na₃O₇/C₆H₈O₇)  0-15% Carboxymethylcellulose 0-2% Polymers (e.g.maleic/acrylic acid copolymer, PVP, 1-6% PEG) Enzymes (calculated aspure enzyme protein) 0.0001-0.1%   Minor ingredients (e.g. sudssuppressors, perfume) 0-5%

[0268] 3) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Linear alkylbenzenesulfonate(calculated as acid) 5-9% Alcohol ethoxylate (e.g. C₁₂₋₁₅ alcohol, 7 EO) 7-14% Soap as fatty acid (e.g. C₁₆₋₂₂ fatty acid) 1-3% Sodium carbonate(as Na₂CO₃) 10-17% Soluble silicate (as Na₂O, 2SiO₂) 3-9% Zeolite (asNaAlSiO₄) 23-33% Sodium sulfate (as Na₂SO4) 0-4% Sodium perborate (asNaBO₃.H₂O)  8-16% TAED 2-8% Phosphonate (e.g. EDTMPA) 0-1%Carboxymethylcellulose 0-2% Polymers (e.g. maleic/acrylic acidcopolymer, PVP, 0-3% PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1%   Minor ingredients (e.g. suds suppressors, perfume, 0-5%optical brightener)

[0269] 4) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Linear alkylbenzenesulfonate(calculated as acid)  8-12% Alcohol ethoxylate (e.g. C₁₂₋₁₅ alcohol, 7EO) 10-25% Sodium carbonate (as Na₂CO₃) 14-22% Soluble silicate (asNa₂O, 2SiO₂) 1-5% Zeolite (as NaAlSiO₄) 25-35% Sodium sulfate (asNa₂SO₄)  0-10% Carboxymethylcellulose 0-2% Polymers (e.g. maleic/acrylicacid copolymer, PVP, 1-3% PEG) Enzymes (calculated as pure enzymeprotein) 0.0001-0.1%   Minor ingredients (e.g. suds suppressors,perfume) 0-5%

[0270] 5) An aqueous liquid detergent composition comprising Linearalkylbenzenesulfonate (calculated as acid) 15-21% Alcohol ethoxylate(e.g. C₁₂₋₁₅ alcohol, 7 EO or 12-18% C_(12-15 alcohol, 5 EO)) Soap asfatty acid (e.g. oleic acid)  3-13% Alkenylsuccinic acid (C₁₂₋₁₄)  0-13%Aminoethanol  8-18% Citric acid 2-8% Phosphonate 0-3% Polymers (e.g.PVP, PEG) 0-3% Borate (as B₄O₇ ²⁻) 0-2% Ethanol 0-3% Propylene glycol 8-14% Enzymes (calculated as pure enzyme protein) 0.0001-0.1%   Minoringredients (e.g. dispersants, suds 0-5% suppressors, perfume, opticalbrightener)

[0271] 6) An aqueous structured liquid detergent composition comprisingLinear alkylbenzenesulfonate (calculated as acid) 15-21% Alcoholethoxylate (e.g. C₁₂₋₁₅ alcohol, 7 EO, or 3-9% C₁₂₋₁₅ alcohol, 5 EO)Soap as fatty acid (e.g. oleic acid)  3-10% Zeolite (as NaAlSiO₄) 14-22%Potassium citrate  9-18% Borate (as B₄O₇ ²⁻) 0-2% Carboxybethylcellulose0-2% Polymers (e.g. PEG, PVP) 0-3% Anchoring polymers such as, e.g.,lauryl 0-3% methacrylate/acrylic acid copolymer; molar ratio 25:1; MW3800 Glycerol 0-5% Enzymes (calculated as pure enzyme protein)0.0001-0.1%   Minor ingredients (e.g. dispersants, suds 0-5%suppressors, perfume, optical brighteners)

[0272] 7) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Fatty alcohol sulfate  5-10%Ethoxylated fatty acid monoethanolamide 3-9% Soap as fatty acid 0-3%Sodium carbonate (as Na₂CO₃)  5-10% Soluble silicate (as Na₂O, 2SiO₂)1-4% Zeolite (as NaAlSiO₄) 20-40% Sodium sulfate (as Na₂SO₄) 2-8% Sodiumperborate (as NaBO₃.H₂O) 12-18% TAED 2-7% Polymers (e.g. maleic/acrylicacid copolymer, PEG) 1-5% Enzymes (calculated as pure enzyme protein)0.0001-0.1%   Minor ingredients (e.g. optical brightener, suds 0-5%suppressors, perfume)

[0273] 8) A detergent composition formulated as a granulate comprisingLinear alkylbenzenesulfonate (calculated as acid)  8-14% Ethoxylatedfatty acid monoethanolamide  5-11% Soap as fatty acid 0-3% Sodiumcarbonate (as Na₂CO₃)  4-10% Soluble silicate (as Na₂O, 2SiO₂) 1-4%Zeolite (as NaAlSiO₄) 30-50% Sodium sulfate (as Na₂SO₄)  3-11% Sodiumcitrate (as C₆H₅Na₃O₇)  5-12% Polymers (e.g. PVP, maleic/acrylic acidcopolymer, 1-5% PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1%   Minor ingredients (e.g. suds suppressors, perfume) 0-5%

[0274] 9) A detergent composition formulated as a granulate comprisingLinear alkylbenzenesulfonate (calculated as acid)  6-12% Nonionicsurfactant 1-4% Soap as fatty acid 2-6% Sodium carbonate (as Na₂CO₃)14-22% Zeolite (as NaAlSiO₄) 18-32% Sodium sulfate (as Na₂SO₄)  5-20%Sodium citrate (as C₆H₅Na₃O₇) 3-8% Sodium perborate (as NaBO₃.H₂O) 4-9%Bleach activator (e.g. NOBS or TAED) 1-5% Carboxymethylcellulose 0-2%Polymers (e.g. polycarboxylate or PEG) 1-5% Enzymes (calculated as pureenzyme protein) 0.0001-0.1%   Minor ingredients (e.g. opticalbrightener, perfume) 0-5%

[0275] 10) An aqueous liquid detergent composition comprising Linearalkylbenzenesulfonate (calculated as acid) 15-23% Alcohol ethoxysulfate(e.g. C₁₂₋₁₅ alcohol, 2-3 EO)  8-15% Alcohol ethoxylate (e.g. C₁₂₋₁₅alcohol, 7 EO, or 3-9% C₁₂₋₁₅ alcohol, 5 EO) Soap as fatty acid (e.g.lauric acid) 0-3% Aminoethanol 1-5% Sodium citrate  5-10% Hydrotrope(e.g. sodium toluene sulfonate) 2-6% Borate (as B₄O₇ ²⁻) 0-2%Carboxymethylcellulose 0-1% Ethanol 1-3% Propylene glycol 2-5% Enzymes(calculated as pure enzyme protein) 0.0001-0.1%   Minor ingredients(e.g. polymers, dispersants, 0-5% perfume, optical brighteners)

[0276] 11) An aqueous liquid detergent composition comprising Linearalkylbenzenesulfonate (calculated as acid) 20-32% Alcohol ethoxylate(e.g. C₁₂₋₁₅ alcohol, 7 EO, or  6-12% C_(12-15 alcohol, 5 EO))Aminoethanol 2-6% Citric acid  8-14% Borate (as B₄O₇ ²⁻) 1-3% Polymer(e.g. maleic/acrylic acid copolymer, 0-3% anchoring polymer such as,e.g., lauryl methacrylate/acrylic acid copolymer) Glycerol 3-8% Enzymes(calculated as pure enzyme protein) 0.0001-0.1%   Minor ingredients(e.g. hydrotropes, dispersants, 0-5% perfume, optical brighteners)

[0277] 12) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising Anionic surfactant (linearalkylbenzenesulfonate, 25-40% alkyl sulfate, alpha-olefinsulfonate,alpha-sulfo fatty acid methyl esters, alkanesulfonates, soap) Nonionicsurfactant (e.g. alcohol ethoxylate)  1-10% Sodium carbonate (as Na₂CO₃) 8-25% Soluble silicates (as Na₂O, 2SiO₂)  5-15% Sodium sulfate (asNa₂SO₄) 0-5% Zeolite (as NaAlSiO₄) 15-28% Sodium perborate (asNaBO₃.4H₂O)  0-20% Bleach activator (TAED or NOBS) 0-5% Enzymes(calculated as pure enzyme protein) 0.0001-0.1%   Minor ingredients(e.g. perfume, optical brighteners) 0-3%

[0278] 13) Detergent formulations as described in 1)-12) wherein all orpart of the linear alkylbenzenesulfonate is replaced by (C₁₂-C₁₈) alkylsulfate.

[0279] 14) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising (C₁₂-C₁₈) alkyl sulfate 9-15% Alcohol ethoxylate 3-6% Polyhydroxy alkyl fatty acid amide 1-5%Zeolite (as NaAlSiO₄) 10-20% Layered disilicate (e.g. SK56 from Hoechst)10-20% Sodium carbonate (as Na₂CO₃)  3-12% Soluble silicate (as Na₂O,2SiO₂) 0-6% Sodium citrate 4-8% Sodium percarbonate 13-22% TAED 3-8%Polymers (e.g. polycarboxylates and PVP) 0-5% Enzymes (calculated aspure enzyme protein) 0.0001-0.1%   Minor ingredients (e.g. opticalbrightener, photo 0-5% bleach, perfume, suds suppressors)

[0280] 15) A detergent composition formulated as a granulate having abulk density of at least 600 g/l comprising (C₁₂-C₁₈) alkyl sulfate 4-8%Alcohol ethoxylate 11-15% Soap 1-4% Zeolite MAP or zeolite A 35-45%Sodium carbonate (as Na₂CO₃) 2-8% Soluble silicate (as Na₂O, 2SiO₂) 0-4%Sodium percarbonate 13-22% TAED 1-8% Carboxymethyl cellulose 0-3%Polymers (e.g. polycarboxylates and PVP) 0-3% Enzymes (calculated aspure enzyme protein) 0.0001-0.1%   Minor ingredients (e.g. opticalbrightener, 0-3% phosphonate, perfume)

[0281] 16) Detergent formulations as described in 1)-15) which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

[0282] 17) Detergent compositions as described in 1), 3), 7), 9) and 12)wherein perborate is replaced by percarbonate.

[0283] 18) Detergent compositions as described in 1), 3), 7), 9), 12),14) and 15) which additionally contain a manganese catalyst. Themanganese catalyst may, e.g., be one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching”, Nature369, 1994, pp. 637-639.

[0284] 19) Detergent composition formulated as a nonaqueous detergentliquid comprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g. phosphate), enzymeand alkali. The detergent may also comprise anionic surfactant and/or ableach system.

[0285] Particular forms of dishwashing detergent compositions within thescope of the invention include:

[0286] 1) Powder Automatic Dishwashing Composition Nonionic surfactant0.4-2.5% Sodium metasilicate  0-20% Sodium disilicate  3-20% Sodiumtriphosphate 20-40% Sodium carbonate  0-20% Sodium perborate 2-9%Tetraacetylethylenediamine (TAED) 1-4% Sodium sulphate  5-33% Enzymes0.0001-0.1%  

[0287] 2) Powder Automatic Dishwashing Composition Nonionic surfactant(e.g. alcohol ethoxylate) 1-2% Sodium disilicate  2-30% Sodium carbonate10-50% Sodium phosphonate 0-5% Trisodium citrate dihydrate  9-30%Nitrilotrisodium acetate (NTA)  0-20% Sodium perborate monohydrate 5-10% Tetraacetylethylenediamine (TAED) 1-2% Polyacrylate polymer (e.g.maleic acid/acrylic acid  6-25% copolymer) Enzymes 0.0001-0.1%   Perfume0.1-0.5% Water  5-10   

[0288] 3) Powder Automatic Dishwashing Composition Nonionic surfactant0.5-2.0% Sodium disilicate 25-40% Sodium citrate 30-55% Sodium carbonate 0-29% Sodium bicarbonate  0-20% Sodium perborate monohydrate  0-15%Tetraacetylethylenediamine (TAED) 0-6% Maleic acid/acrylic acidcopolymer 0-5% Clay 1-3% Poly(amino acids)  0-20% Sodium polyacrylate0-8% Enzymes 0.0001-0.1%  

[0289] 4) Powder Automatic Dishwashing Composition Nonionic surfactant1-2% Zeolite MAP 15-42% Sodium disilicate 30-34% Sodium citrate  0-12%Sodium carbonate  0-20% Sodium perborate monohydrate  7-15%Tetraacetylethylenediamine (TAED) 0-3% Polymer 0-4% Maleic acid/acrylicacid copolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes0.0001-0.1%   Sodium sulphate Balance

[0290] 5) Powder Automatic Dishwashing Composition Nonionic surfactant1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate12-20% Monopersulphate (2 KHSO₅.KHSO₄.K₂SO₄) 15-21% Bleach stabilizer0.1-2%   Maleic acid/acrylic acid copolymer 0-6%Diethylenetriaminepentaacetate, pentasodium salt   0-2.5% Enzymes0.0001-0.1%   Sodium sulphate, water Balance

[0291] 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANINGSURFACTANT SYSTEM Nonionic surfactant   0-1.5% Octadecyl dimethylamineN-oxide dihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyldimethylamine 0-4% N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5%(hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis(hydroxyethyl)amine N-oxide anhydrous C₁₃-C₁₅ alkyl ethoxysulfate withan average degree  0-10% of ethoxylation of 3 C₁₂-C₁₅ alkylethoxysulfate with an average degree 0-5% of ethoxylation of 3 C₁₃-C₁₅ethoxylated alcohol with an average degree 0-5% of ethoxylation of 12 Ablend of C₁₂-C₁₅ ethoxylated alcohols with an   0-6.5% average degree ofethoxylation of 9 A blend of C₁₃-C₁₅ ethoxylated alcohols with an 0-4%average degree of ethoxylation of 30 Sodium disilicate  0-33% Sodiumtripolyphosphate  0-46% Sodium citrate  0-28% Citric acid  0-29% Sodiumcarbonate  0-20% Sodium perborate monohydrate   0-11.5%Tetraacetylethylenediamine (TAED) 0-4% Maleic acid/acrylic acidcopolymer   0-7.5% Sodium sulphate   0-12.5% Enzymes 0.0001-0.1%  

[0292] 7) Non-Aqueous Liquid Automatic Dishwashing Composition Liquidnonionic surfactant (e.g. alcohol ethoxylates)  2.0-10.0% Alkali metalsilicate  3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrierselected from higher glycols, 25.0-45.0% polyglycols, polyoxides,glycolethers Stabilizer (e.g. a partial ester of phosphoric acid and0.5-7.0% a C_(16-C18) alkanol) Foam suppressor (e.g. silicone)   0-1.5%Enzymes 0.0001-0.1%  

[0293] 8) Non-Aqueous Liquid Dishwashing Composition Liquid nonionicsurfactant (e.g. alcohol ethoxylates)  2.0-10.0% Sodium silicate 3.0-15.0% Alkali metal carbonate  7.0-20.0% Sodium citrate 0.0-1.5%Stabilizing system (e.g. mixtures of finely divided 0.5-7.0% siliconeand low molecular weight dialkyl polyglycol ethers) Low molecule weightpolyacrylate polymer  5.0-15.0% Clay gel thickener (e.g. bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes0.0001-0.1%   Liquid carrier selected from higher lycols, Balancepolyglycols, polyoxides and glycol ethers

[0294] 9) Thixotropic Liquid Automatic Dishwashing CompositionC_(12-C14) fatty acid   0-0.5% Block co-polymer surfactant  1.5-15.0%Sodium citrate  0-12% Sodium tripolyphosphate  0-15% Sodium carbonate0-8% Aluminum tristearate   0-0.1% Sodium cumene sulphonate   0-1.7%Polyacrylate thickener 1.32-2.5%  Sodium polyacrylate 2.4-6.0% Boricacid   0-4.0% Sodium formate   0-0.45% Calcium formate   0-0.2% Sodiumn-decydiphenyl oxide disulphonate   0-4.0% Monoethanol amine (MEA)  0-1.86% Sodium hydroxide (50%) 1.9-9.3% 1,2-Propanediol   0-9.4%Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance

[0295] 10) Liquid Automatic Dishwashing Composition Alcohol ethoxylate 0-20% Fatty acid ester sulphonate  0-30% Sodium dodecyl sulphate  0-20%Alkyl polyglycoside  0-21% Oleic acid  0-10% Sodium disilicatemonohydrate 18-33% Sodium citrate dihydrate 18-33% Sodium stearate  0-2.5% Sodium perborate monohydrate  0-13% Tetraacetylethylenediamine(TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8% Enzymes0.0001-0.1%  

[0296] 11) Liquid Automatic Dishwashing Composition Containing ProtectedBleach Particles Sodium silicate  5-10% Tetrapotassium pyrophosphate15-25% Sodium triphosphate 0-2% Potassium carbonate 4-8% Protectedbleach particles, e.g. chlorine  5-10% Polymeric thickener 0.7-1.5%Potassium hydroxide 0-2% Enzymes 0.0001-0.1%   Water Balance

[0297] 11) Automatic dishwashing compositions as described in 1), 2),3), 4), 6) and 10), wherein perborate is replaced by percarbonate.

[0298] 12) Automatic dishwashing compositions as described in 1)-6)which additionally contain a manganese catalyst. The manganese catalystmay, e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp.637-639.

[0299] An α-amylase variant of the invention may be incorporated inconcentrations conventionally employed in detergents. It is at presentcontemplated that, in the detergent composition of the invention, theα-amylase variant may be added in an amount corresponding to 0.00001-1mg (calculated as pure enzyme protein) of α-amylase per liter ofwash/dishwash liquor.

[0300] The present invention is further described with reference to theappended drawing, in which:

[0301] FIG. 1 is an alignment of the amino acid sequences of four parentα-amylases in the context of the invention. The numbers on the extremeleft designate the respective amino acid sequences as follows:

[0302] 1: the amino acid sequence shown in SEQ ID No. 1;

[0303] 2: the amino acid sequence shown in SEQ ID No. 2;

[0304] 3: the amino acid sequence shown in SEQ ID No. 3; and

[0305] 4: the amino acid sequence shown in SEQ ID No. 7.

[0306] The numbers on the extreme right of the figure give the runningtotal number of amino acids for each of the sequences in question. Itshould be noted that for the sequence numbered 3 (corresponding to theamino acid sequence shown in SEQ ID No. 3), the alignment results in“gaps” at the positions corresponding to amino acid No. 1 and amino acidNo. 175, respectively, in the sequences numbered 1 (SEQ ID No. 1), 2(SEQ ID No. 2) and 4 (SEQ ID No. 7).

[0307] FIG. 2 is a restriction map of plasmid pTVB106.

[0308] FIG. 3 is a restriction map of plasmid pPM103.

[0309] FIG. 4 is a restriction map of plasmid pTVB112.

[0310] FIG. 5 is a restriction map of plasmid pTVB114.

EXPERIMENTAL SECTION

[0311] The preparation, purification and sequencing of the parentα-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQID No. 2 (from Bacillus strains NCIB 12512 and NCIB 12513, respectively)is described in WO 95/26397. The pi values and molecular weights ofthese two parent α-amylases (given in WO 95/26397) are as follows:

[0312] SEQ ID No. 1: pi about 8.8-9.0 (determined by isoelectricfocusing on LKB Ampholine□ PAG plates); molecular weight approximately55 kD (determined by SDS-PAGE).

[0313] SEQ ID No. 2: pi about 5.8 (determined by isoelectric focusing onLKB Ampholine□ PAG plates); molecular weight approximately 55 kD(determined by SDS-PAGE).

[0314] Purification of α-Amylase Variants of the Invention

[0315] The construction and expression of variants according to theinvention is described in Example 2, below. The purification of variantsof the invention is illustrated here with reference to variants of theamino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2,respectively:

[0316] Purification of SEQ ID No. 1 variants (pI approx. 9.0): Thefermentation liquid containing the expressed α-amylase variant isfiltered, and ammonium sulfate is added to a concentration of 15% ofsaturation. The liquid is then applied onto a hydrophobic column(Toyopearl butyl/TOSOH). The column is washed with 20 mMdimethyl-glutaric acid buffer, pH 7.0. The α-amylase is bound verytightly, and is eluted with 25% w/w 2-propanol in 20 mM dimethylglutaricacid buffer, pH 7.0. After elution, the 2-propanol is removed byevaporation and the concentrate is applied onto a cation exchanger(S-Sepharose□ FF, Pharmacia, Sweden) equilibrated with 20 mMdimethylglutaric acid buffer, pH 6.0.

[0317] The amylase is eluted using a linear gradient of 0-250 mM NaCl inthe same buffer. After dialysis against 10 mM borate/KCl buffer, pH 8.0,the sample is adjusted to pH 9.6 and applied to an anion exchanger(Q-Sepharose[ ] FF, Pharmacia) equilibrated with 10 mM borate/KClbuffer, pH 9.6. The amylase is eluted using a linear gradient of 0-250mM NaCl. The pH is adjusted to 7.5. The α-amylase is pure as judged byrSDS-PAGE. All buffers contain 2 mM CaCl₂ in order to stabilize theamylase.

[0318] Purification of SEQ ID No. 2 variants (pI approx. 5.8): Thefermentation liquid containing the expressed α-amylase variant isfiltered, and ammonium sulfate is added to a concentration of 15% ofsaturation. The liquid is then applied onto a hydrophobic column(Toyopearl butyl/TOSOH). The bound amylase is eluted with a lineargradient of 15%-0% w/w ammonium sulfate in 10 mM Tris buffer, pH 8.0.After dialysis of the eluate against 10 mM borate/KCl buffer, pH 8.0,the liquid is adjusted to pH 9.6 and applied onto an anion exchanger(Q-Sepharose□ FF, Pharmacia) equilibrated with the same buffer. Theamylase is step-eluted using 150 mM NaCl.

[0319] After elution the amylase sample is dialyzed against the samebuffer, pH 8.0, in order to remove the NaCl. After dialysis, the pH isadjusted to 9.6 and the amylase is bound once more onto the anionexchanger. The amylase is eluted using a linear gradient of 0-250 mMNaCl. The pH is adjusted to 7.5. The amylase is pure as judged byrSDS-PAGE. All buffers contain 2 mM CaCl₂ in order to stabilize theamylase.

[0320] Determination of α-Amylase Activity

[0321] α-Amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer which has been mixed with bovine serum albumin and abuffer substance and tabletted.

[0322] For the determination of every single measurement one tablet issuspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mMacetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pHadjusted to the value of interest with NaOH). The test is performed in awater bath at the temperature of interest. The α-amylase to be tested isdiluted in ×ml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the α-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the α-amylaseactivity.

[0323] It is important that the measured 620 nm absorbance after 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion.

[0324] Under a specified set of conditions (temp., pH, reaction time,buffer conditions) 1 mg of a given α-amylase will hydrolyze a certainamount of substrate and a blue color will be produced. The colorintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure α-amylaseprotein) of the α-amylase in question under the given set of conditions.Thus testing different α-amylases of interest (including a referenceα-amylase, in this case the parent α-amylase in question) underidentical conditions, the specific activity of each of the α-amylases ata given temperature and at a given pH can be compared directly, and theratio of the specific activity of each of the α-amylases of interestrelative to the specific activity of the reference α-amylase can bedetermined.

[0325] Mini Dishwashing Assay

[0326] The following mini dishwashing assay was used: A suspension ofstarchy material was boiled and cooled to 20□C. The cooled starchsuspension was applied on small, individually identified glass plates(approx. 2×2 cm) and dried at a temperature of ca. 140□C in a dryingcabinet. The individual plates were then weighed. For assay purposes, asolution of standard European-type automatic dishwashing detergent (5g/l) having a temperature of 55□C was prepared. The detergent wasallowed a dissolution time of 1 minute, after which the α-amylase inquestion was added to the detergent solution (contained in a beakerequipped with magnetic stirring) so as to give an enzyme concentrationof 0.5 mg/l. At the same time, the weighed glass plates, held in smallsupporting clamps, were immersed in a substantially vertical position inthe α-amylase/detergent solution, which was then stirred for 15 minutesat 55□C. The glass plates were then removed from the α-amylase/detergentsolution, rinsed with distilled water, dried at 60□C in a drying cabinetand re-weighed. The performance of the α-amylase in question [expressedas an index relative to a chosen reference α-amylase (index 100)—in theexample below (Example 1) the parent α-amylase having the amino acidsequence shown in SEQ ID No. 1] was then determined from the differencein weight of the glass plates before and after treatment, as follows:${Index} = {\frac{{weight}\quad {loss}\quad {for}\quad {plate}\quad {treated}\quad {with}\quad \alpha \text{-}{amylase}}{{weight}\quad {loss}\quad {for}\quad {plate}\quad {treated}\quad {with}\quad {reference}}\quad \bullet \quad 100}$

[0327] The following examples further illustrate the present invention.They are not intended to be in any way limiting to the scope of theinvention as claimed.

Example 1

[0328] Mini Dishwashing Test of Variants of Parent α-Amylase Having TheAmino Acid Sequence Shown in SEQ ID No. 1

[0329] The above-described mini dishwashing test was performed at pH10.5 with the parent α-amylase having the amino acid sequence shown inSEQ ID No. 1 and the following variants thereof (the construction andpurification of which is described below): T183*+G184*; Y243F; andK269R. The test gave the following results: Parent (SEQ ID No. 1) Index:100 T183* + G184* Index: 120 Y243F Index: 120 K269R Index: 131

[0330] It is apparent that the each of the tested variants T183*+G184*(which exhibits, inter alia, higher thermal stability than the parentα-amylase), Y243F (which exhibits lower calcium ion dependency than theparent α-amylase) and K269R (which exhibits lower calcium ion dependencyand higher stability at high pH than the parent α-amylase) exhibitssignificantly improved dishwashing performance relative to the parentα-amylase.

Example 2

[0331] Construction of Variants of the Parent α-Amylases Having theAmino Acid Sequences Shown in SEQ ID No. 1 and SEQ ID No. 2,Respectively

[0332] Primers: DNA primers employed in the construction of variants asdescribed below include the following [all DNA primers are written inthe direction from 5′ to 3′ (left to right); P denotes a 5′ phosphate]:#7113: GCT GCG GTG ACC TCT TTA AAA AAT AAC GGC Y296: CC ACC GCT ATT AGATGC ATT GTA C #6779: CTT ACG TAT GCA GAC GTC GAT ATG GAT CAC CC #6778: GATC CAT ATC GAC GTC TGC ATA CGT AAG ATA GTC #3811: TT A(C/G)G GGC AAGGCC TGG GAC TGG #7449: C CCA GGC CTT GCC C(C/G)T AAA TTT ATA TAT TTT GTTTTG #3810: G GTT TCG GTT CGA AGG ATT CAC TTC TAC CGC #7450: GCG GTA GAAGTG AAT CCT TCG AAC CGA AAC CAG B1: GGT ACT ATC GTA ACA ATG GCC GAT TGCTGA CGC TGT TAT TTG C #6616: P CTG TGA CTG GTG AGT ACT CAA CCA AGT C#8573: CTA CTT CCC AAT CCC AAG CTT TAC CTC GGA ATT TG #8569: CAA ATT CCGAGG TAA AGC TTG GGA TTG GGA AGT AG #8570: TTG AAC AAC CGT TCC ATT AAGAAG

[0333] A: Construction of Variants of the Parent α-Amylase Having theAmino Acid Sequence Shown in SEQ ID No. 1

[0334] Description of plasmid pTVB106: The parent α-amylase having theamino acid sequence shown in SEQ ID No. 1 and variants thereof areexpressed from a plasmid-borne gene, SF16, shown in FIG. 2. The plasmid,pTVB106, contains an origin of replication obtained from plasmid pUB 110(Gryczan et al., 1978) and the cat gene conferring resistance towardschloramphenicol. Secretion of the amylase is aided by the Termamyl□signal sequence that is fused precisely, i.e. codon No. 1 of the matureprotein, to the gene encoding the parent α-amylase having the nucleotideand amino acid sequence (mature protein) shown in SEQ ID No. 4 and SEQID No. 1, respectively. The Termamyl promoter initiates transcription ofthe gene.

[0335] Plasmid pTVB106 is similar to pDN1528 (see laid-open Danishpatent application No. 1155/94). Some unique restriction sites areindicated on the plasmid map in FIG. 2, including BstBI, BamHI, BstEII,EcoNI, DrdI, AflIII, DraIII, XmaI, SalI and BglII.

[0336] Construction of variant M202T: The PCR overlap extensionmutagenesis method is used to construct this variant (Higuchi et al.,1988). An approximately 350 bp DNA fragment of pTVB106 is amplified in aPCR reaction A using primers #7113 and mutagenic primer#6778. In asimilar PCR reaction B, an approximately 300 bp DNA fragment isamplified using primers Y296 and #6779. The complete DNA fragmentspanning the mutation site (M202) from primer #7113 to primer Y296 isamplified in PCR C using these primers and purified DNA fragments fromreactions A and B.

[0337] PCR C DNA is digested with restriction endonucleases BstEII andAflIII, and the 480 bp fragment is ligated with plasmid pTVB106 digestedwith the same enzymes and transformed into a low-protease andlow-amylase Bacillus subtilis strain (e.g. strain SHA273 mentioned in WO92/11357).

[0338] Other M202 variants are constructed in a similar manner.

[0339] Construction of variants T183*+G184* and R181*+G182*: The PCRoverlap extension mutagenesis method is used to construct these variants(Higuchi et al., 1988). The mutagenic oligonucleotides are synthesizedusing a mixture (equal parts) of C and G in one position; two differentmutations can therefore be constructed by this procedure. Anapproximately 300 bp DNA fragment of pTVB106 is amplified in a PCRreaction A using primers #7113 and mutagenic primer #7449. In a similarPCR reaction B, an approximately 400 bp DNA fragment is amplified usingprimers Y296 and #3811. The complete DNA fragment spanning the mutationsite (amino acids 181-184) from primer #7113 to primer Y296 is amplifiedin PCR C using these primers and purified DNA fragments from reactions Aand B.

[0340] PCR C DNA is digested with restriction endonucleases BstEII andAflIII and the 480 bp fragment is ligated with plasmid pTVB106 digestedwith the same enzymes and transformed into a low-protease andlow-amylase B. subtilis strain (e.g. strain SHA273 mentioned in WO92/11357). Sequencing of plasmid DNA from these transformants identifiesthe two correct mutations: i.e. R181*+G182* and T183*+G184*.

[0341] Construction of variant R124P: The PCR overlap extensionmutagenesis method is used to construct this variant in a manner similarto the construction of variant M202T (vide supra). PCR reaction A (withprimers #3810 and B1) generates an approximately 500 bp fragment, andPCR reaction B (primers 7450 and Y296) generates an approximately 550 bpfragment. PCR reaction C based on the product of PCR reaction A and Band primers B1 and Y296 is digested with restriction endonucleasesBstEII and AflIII, and the resulting 480 bp fragment spanning amino acidposition 124 is subcloned into pTVB106 digested with the same enzymesand transformed into B. subtilis as previously described.

[0342] Construction of variant R124P+T183*+G184*: For the constructionof the variant combining the R124P and the T183*+G184* mutations, twoEcoNI restriction sites (one located at position 1.774 kb, i.e. betweenthe R124P mutation and the T183*+G184* mutation, and one located atposition 0.146 kb) were utilized. The approximately 1630 bp EcoNIfragment of the pTVB106-like plasmid containing the T183*+G184* mutationwas subcloned into the vector part (approximately 3810 bp DNA fragmentcontaining the origin of replication) of another pTVB106-like plasmidcontaining the R124P mutation digested with the same enzyme.Transformation into Bacillus subtilis was carried out as previouslydescribed.

[0343] Construction of variants G182*+G184*; R181*+T183*; Y243F; K269R;and L351C+M430C: These variants were constructed as follows:

[0344] A specific mutagenesis vector containing a major part of thecoding region for the amino acid sequence shown in SEQ ID No. 1 wasprepared. The important features of this vector (which is denoted pPM103) include an origin of replication derived from the pUC plasmid, thecat gene conferring resistance towards chloramphenicol and aframeshift-mutation-containing version of the bla gene, the wild-typeversion of which normally confers resistance towards ampicillin (amp^(R)phenotype). This mutated version of the bla gene results in an amp^(S)phenotype. The plasmid pPM103 is shown in FIG. 3, and the E. coli originof replication, the 5′-truncated version of the SF16 amylase gene, andori, bla, cat and selected restriction sites are indicated on theplasmid.

[0345] Mutations are introduced in the gene of interest as described byDeng and Nickoloff [Anal. Biochem. 200 (1992), pp. 81-88], except thatplasmids with the “selection primer” (#6616) incorporated are selectedbased on the amp^(R) phenotype of transformed E coli cells harboring aplasmid with a repaired bla gene instead of using the selection byrestriction-enzyme digestion outlined by Deng and Nickoloff. Chemicalsand enzymes used for the mutagenesis were obtained from the Chameleon□mutagenesis kit from Stratagene (catalogue number 200509).

[0346] After verification of the DNA sequence in variant plasmids, thetruncated gene containing the desired alteration is subcloned from thepPM103-like plasmid into pTVB106 as an approximately 1440 bp BstBI-SalIfragment and transformed into Bacillus subtilis for expression of thevariant enzyme.

[0347] For the construction of the pairwise deletion variantG182*+G184*, the following mutagenesis primer was used: P CTC TGT ATCGAC TTC CCA GTC CCA AGC TTT TGT CCT GAA TTT ATA TAT TTT GTT TTG AAG

[0348] For the construction of the pairwise deletion variantR181*+T183*, the following mutagenesis primer was used: P CTC TGT ATCGAC TTC CCA GTC CCA AGC TTT GCC TCC GAA TTT ATA TAT TTT GTT TTG AAG

[0349] For the construction of the substitution variant Y243F, thefollowing mutagenesis primer was used: P ATG TGT AAG CCA ATC GCG AGT AAAGCT AAA TTT TAT ATG TTT CAC TGC ATC

[0350] For the construction of the substitution variant K269R, thefollowing mutagenesis primer was used: P GC ACC AAG GTC ATT TCG CCA GAATTC AGC CAC TG

[0351] For the construction of the pairwise substitution variantL351C+M430C, the following mutagenesis primers were usedsimultaneously: 1) P TGT CAG AAC CAA CGC GTA TGC ACA TGG TTT AAA CCA TTG2) P ACC ACC TGG ACC ATC GCT GCA GAT GGT GGC AAG GCC TGA ATT

[0352] Construction of variant L351C+M430C+T183*+G184*: This variant wasconstructed by combining the L351C+M430C pairwise substitution mutationand the T183*+G184* pairwise deletion mutation by subcloning anapproximately 1430 bp HindIII-AflIII fragment containing L351C+M430Cinto a pTVB106-like plasmid (with the T183*+G184* mutations) digestedwith the same enzymes.

[0353] Construction of variant Y243F+T183*+G184*: This variant wasconstructed by combining the Y243F mutation and the T183*+G184* mutationby subcloning an approximately 1148 bp Drdl fragment containingT183*+G184* into a pTVB106-like plasmid (with the Y243 mutation)digested with the same enzyme.

[0354]Bacillus subtilis transformants were screened for α-amylaseactivity on starch-containing agar plates and the presence of thecorrect mutations was checked by DNA sequencing.

[0355] Construction of variant Y243F+T183*+G184*+L351C+M430C: TheL351C+M430C pairwise substitution mutation was subcloned as anapproximately 470 bp XmaI-SalI fragment into a pTVB106-like vector(containing Y243F+T183*+G184*) digested with the same enzymes.

[0356] Construction of variant Y243F+T183*+G184*+L351C+M430C+Q391E+K444Q: A pPM103-like vector containing the mutationsY243F+T183*+G184*+L351C+M430C was constructed by substituting thetruncated version of SF16 in pPM103 with the approximately 1440 bpBstB1-SalI fragment of the pTVB106-like vector containing the fivemutations in question. The Q391 E and K444Q mutations were introducedsimultaneously into the pPM103-like vector (containingY243F+T183*+G184*+L351C+M430C) by the use of the following twomutagenesis primers in a manner similar to the previously describedmutagenesis on pPM103: P GGC AAA AGT TTG ACG TGC CTC GAG AAG AGG GTC TATP TTG TCC CGC TTT ATT CTG GCC AAC ATA CAT CCA TTT

[0357] B: Construction of Variants of the Parent α-Amylase Having theAmino Acid Sequence Shown in SEQ ID No. 2

[0358] Description of plasmid pTVB112: Avector, denoted pTVB112, to beused for the expression in B. subtilis of the α-amylase having the aminoacid sequence shown in SEQ ID No. 2 was constructed. This vector is verysimilar to pTVB106 except that the gene encoding the mature α-amylase ofSEQ ID No. 2 is inserted between the PstI and the HindIII sites inpTVB106. Thus, the expression of this α-amylase (SEQ ID No. 2) is alsodirected by the amyL promoter and signal sequence. The plasmid pTVB112is shown in FIG. 4.

[0359] Construction of variant D183*+G184*: The construction of thisvariant was achieved using the PCR overlap extension mutagenesis methodreferred to earlier (vide supra). Primers #8573 and B1 were used in PCRreaction A, and primers #8569 and #8570 were used in PCR reaction B. Thepurified fragments from reaction A and reaction B and primers 1 B and#8570 were used in PCR reaction C, resulting in an approximately 1020 bpDNA fragment. This fragment was digested with restriction endonucleasesPstI and MluI, and subcloned into the expression vector and transformedinto B. subtilis.

[0360] Construction of further variants: By analogy with theconstruction (vide supra) of the plasmid pPM103 used in the productionof mutants of the amino acid sequence of SEQ ID No. 1, a plasmid(denoted pTVB114; shown in FIG. 5) was constructed for the continuedmutagenesis on variant D183*+G184* (SEQ.ID No. 2). Mutations wereintroduced in pTVB114 (SEQ ID No. 2; D183*+G184*) in a manner similar tothat for pPM103 (SEQ ID No. 1).

[0361] For the construction of the pairwise deletion variantsR181*+Dl83* and R181*+G182*, it was chosen to alter the flanking aminoacids in the variant D183*+G184* instead of deleting the specified aminoacids in the wild type gene for SEQ ID No. 2. The following mutagenesisprimer was used for the mutagenesis with pTVB114 as template: PCC CAATCC CAA GCT TTA CCA (T/C)CG AAC TTG TAG ATA CG

[0362] The presence of a mixture of two bases (T/C) at one positionallows for the presence of two different deletion flanking amino acidbased on one mutagenesis primer. DNA sequencing of the resultingplasmids verifies the presence of either the one or the other mutation.The mutated gene of interest is subcloned as a Pstl-DraIII fragment intopTVB112 digested with the same enzymes and transformed into B. subtilis.

[0363] For the construction of G182*+G184* and R181*+G184*, thefollowing mutagenesis primer was used with pTVB114 as template: PCC CAATCC CAA GCT TTA TCT C(C/G)G AAC TTG TAG ATA CG

[0364] As before, the presence of a mixture of two bases (C/G) at oneposition allows for the presence of two different deletion flankingamino acid based on one mutagenesis primer. DNA sequencing of theresulting plasmids verifies the presence of either the one or the othermutation. The mutated gene of interest is subcloned as a Pstl-DraIIIfragment into pTVB112 digested with the same enzymes and transformedinto B. subtilis. For the construction of D183* + G184* + M202L thefollowing mutagenesis primer was used: PGA TCC ATA TCG ACG TCT GCA TACAGT AAA TAA TC For the construction of D183* + G184* + M202I thefollowing mutagenesis primer was used: PGA TCC ATA TCG ACG TCT GCA TAAATT AAA TAA TC

Example 3

[0365] Determination of Oxidation Stability of M202 SubstitutionVariants of the Parent α-Amylases Having the Amino Acid Sequences Shownin SEQ ID No. 1 and SEQ ID No. 2

[0366] A: Oxidation Stability of Variants of the Sequence in SEQ ID No.1

[0367] The measurements were made using solutions of the respectivevariants in 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH), pH 9.0, to which hydrogen peroxide wasadded (at time t=0) to give a final concentration of 200 mM H₂O₂. Thesolutions were then incubated at 40□C in a water bath.

[0368] After incubation for 5, 10, 15 and 20 minutes after addition ofhydrogen peroxide, the residual α-amylase activity was measured usingthe Phadebas assay described above. The residual activity in the sampleswas measured using 50 mM Britton-Robinson buffer, pH 7.3, at 37□C (seeNovo analytical publication AF207-1/1, available on request from NovoNordisk A/S). The decline in activity was measured relative to acorresponding reference solution of the same enzyme at 0 minutes whichwas not incubated with hydrogen peroxide (100% activity).

[0369] The percentage of initial activity as a function of time is shownin the table below for the parent enzyme (SEQ ID No. 1) and for thevariants in question. % Activity after incubation for (minutes) Variant0 5 10 15 20 M202L 100 90 72 58 27 M202F 100 100 87 71 43 M202A 100 9982 64 30 M202I 100 91 75 59 28 M202T 100 87 65 49 20 M202V 100 100 87 7443 M202S 100 100 85 68 34 Parent 100 51 26 13 2

[0370] All the M202 substitution variants tested clearly exhibitsignificantly improved stability towards oxidation relative to theparent α-amylase (SEQ ID No. 1).

[0371] B: Oxidation Stability of Variants of the Sequence in SEQ ID No.2

[0372] Measurements were made as described above using the parentα-amylase in question (SEQ ID No. 2), the variant M202L+D183*+G184*(designated L in the table below) and the variant M2021+D183*+G184*(designated I in the table below), respectively. In this case,incubation times (after addition of hydrogen peroxide) of 5, 10, 15 and30 minutes were employed. As in the table above, the percentage ofinitial activity as a function of time is shown in the table below forthe parent enzyme and for the variants in question. P% Activity afterincubation for (minutes) Variant 0 5 10 15 30 L 100 91 85 71 43 I 100 8161 44 18 Parent 100 56 26 14 4

[0373] The two “substitution+pairwise deletion” variants tested (whichboth comprise an M202 substitution) clearly exhibit significantlyimproved stability towards oxidation relative to the parent α-amylase(SEQ ID No. 2).

Example 4

[0374] Determination of Thermal Stability of Variants of the Parentα-Amylases Having the Amino Acid Sequences Shown in SEQ ID No. 1 and SEQID No. 2

[0375] A: Thermal Stability of Pairwise Deletion Variants of theSequence in SEQ ID No. 1

[0376] Measurements were made using solutions of the respective variantsin 50 mM Britton-Robinson buffer (vide supra), pH 9.0. The solutionswere incubated at 650C in a water bath, and samples were withdrawn afterincubation for the indicated periods of time. The residual α-amylaseactivity of each withdrawn sample was measured using the Phadebas assay,as described above. The decline in activity was measured relative to acorresponding reference solution of the same enzyme at 0 minutes whichwas not incubated (100% activity).

[0377] The percentage of initial activity as a function of time is shownin the table below for the parent enzyme (SEQ ID No. 1) and for thefollowing pairwise deletion variants in question:

[0378] Variant 1: R181*+G182*

[0379] Variant 2: R181*+T183*

[0380] Variant 3: G182*+G184*

[0381] Variant 4: T183*+G184*

[0382] Variant 5: T183*+G184*+R124P % Activity after incubation for(minutes) Variant 0 5 10 15 30 45 60 1 100 81 66 49 24 14 8 2 100 80 5339 17 8 3 3 100 64 40 28 10 4 2 4 100 64 43 34 20 8 5 5 100 78 73 66 5747 38 Parent 100 13 2 0 0 0 0

[0383] It is apparent that all of the pairwise deletion variants testedexhibit significantly improved thermal stability relative to the parentα-amylase (SEQ ID No. 1), and that the thermal stability of Variant 5,which in addition to the pairwise deletion mutation of Variant 4comprises the substitution R124P, is markedly higher than that of theother variants. Since calorimetric results for the substitution variantR124P (comprising only the substitution R124P) reveal an approximately7° C. thermostabilization thereof relative to the parent α-amylase, itappears that the thermostabilizing effects of the mutation R124P and thepairwise deletion, respectively, reinforce each other.

[0384] B: Thermal Stability of Pairwise Deletion Variants of theSequence in SEQ ID No. 2

[0385] Corresponding measurements were made for the parent enzyme (SEQID No. 2) and for the following pairwise deletion variants:

[0386] Variant A: D183*+G184*

[0387] Variant B: R181*+G182*

[0388] Variant C: G182*+G184* % Activity after incubation for (minutes)Variant 0 5 10 15 30 A 100 87 71 63 30 B 100 113 85 76 58 C 100 99 76 6234 Parent 100 72 55 44 18

[0389] Again, it is apparent that the pairwise deletion variants inquestion exhibit significantly improved thermal stability relative tothe parent α-amylase (SEQ ID No. 2).

[0390] C: Thermal Stability of a Multi-Combination Variant of theSequence in SEQ ID No. 1

[0391] Corresponding comparative measurements were also made for thefollowing variants of the amino acid sequence shown in SEQ ID No. 1:

[0392] Variant 4: T183*+G184*

[0393] Variant 6: L351C+M430C

[0394] Variant 7: Y243F

[0395] Variant 8: Q391 E+K444Q

[0396] Variant 9: T183*+G184*+L351C+M430C+Y243F+Q391E+K444Q % Activityafter incubation for (minutes) Variant 0 5 10 15 30 4 100 66 41 22 7 6100 87 73 65 43 7 100 14 2 1 0 8 100 69 46 31 14 9 100 92 93 89 82

[0397] Again, it appears that the thermostabilizing effect of multiplemutations, each of which has a thermostabilizing effect, is—at leastqualitatively—cumulative.

Example 5

[0398] Calcium-Binding Affinity of α-Amylase Variants of the Invention

[0399] Unfolding of amylases by exposure to heat or to denaturants suchas guanidine hydrochloride is accompanied by a decrease in fluorescence.Loss of calcium ions leads to unfolding, and the affinity of a series ofα-amylases for calcium can be measured by fluorescence measurementsbefore and after incubation of each α-amylase (e.g. at a concentrationof 10 μg/ml) in a buffer (e.g. 50 mM HEPES, pH 7) with differentconcentrations of calcium (e.g. in the range of 1 μM-100 mM) or of EGTA(e.g. in the range of 1-1000 μM)[EGTA=1,2-di(2-aminoethoxy)ethane-N,N,N′,N′-tetraacetic acid] for asufficiently long period of time (such as 22 hours at 55□C).

[0400] The measured fluorescence F is composed of contributions form thefolded and unfolded forms of the enzyme. The following equation can bederived to describe the dependence of F on calcium concentration ([Ca]):

F=[Ca]/(K _(diss)+[Ca])(α_(N)−β_(N)log([Ca]))+K _(diss)/(K_(diss)+[Ca])(α_(U)−β_(U)log([Ca]))

[0401] where α_(N) is the fluorescence of the native (folded) form ofthe enzyme, β_(N) is the linear dependence of α_(N) on the logarithm ofthe calcium concentration (as observed experimentally), α_(U) is thefluorescence of the unfolded form and β_(U) is the linear dependence ofα_(U) on the logarithm of the calcium concentration. K_(diss) is theapparent calcium-binding constant for an equilibrium process as follows:${N\quad —\quad {Ca}\quad \overset{K_{diss}}{\bullet}\quad U} + {{Ca}\quad \left( {{N = {{native}\quad {enzyme}}};{U = {{unfolded}\quad {enzyme}}}} \right)}$

[0402] In fact, unfolding proceeds extremely slowly and is irreversible.The rate of unfolding is a dependent on calcium concentration, and thedependency for a given α-amylase provides a measure of the Ca-bindingaffinity of the enzyme. By defining a standard set of reactionconditions (e.g. 22 hours at 55□C), a meaningful comparison of K_(diss)for different α-amylases can be made. The calcium dissociation curvesfor α-amylases in general can be fitted to the equation above, allowingdetermination of the corresponding values of K_(diss).

[0403] The following values for K_(diss) were obtained for the parentα-amylases having the amino acid sequences shown in SEQ ID No. 1 and SEQID No. 2, and for the indicated α-amylase variants according to theinvention (the parent α-amylase being indicated in parentheses): VariantK_(diss) (mol/l) D183* + G184* (SEQ ID No. 2) 1.2 (±0.5) × 10⁻⁴ L351C +M430C + T183* + G184* 1.7 (±0.5) × 10⁻³ (SEQ ID No. 1) T183* + G184*(SEQ ID No. 1) 4.3 (±0.7) × 10⁻³ SEQ ID No. 2 (parent) 4.2 (±1.2) × 10⁻²SEQ ID No. 1 (parent) 3.5 (±1.1) × 10⁻¹

[0404] It is apparent from the above that the calcium-binding affinityof the latter α-amylolytic enzymes decreases in a downward directionthrough the above table, i.e. that the pairwise deletion variantD183*+G184* (SEQ ID No. 2) binds calcium most strongly (i.e. has thelowest calcium dependency) whilst the parent α-amylase of SEQ ID No. 1binds calcium least strongly (i.e. has the highest calcium dependency).

REFERENCES CITED IN THE SPECIFICATION

[0405] Suzuki et al., the Journal of Biological Chemistry, Vol. 264, No.32, Issue of November 15, pp. 18933-18938 (1989).

[0406] Hudson et al., Practical Immunology, Third edition (1989),Blackwell Scientific Publications.

[0407] Lipman and Pearson (1985) Science 227, 1435.

[0408] Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor, 1989.

[0409] S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981,pp. 1859-1869.

[0410] Matthes et al., The EMBO J. 3, 1984, pp. 801-805.

[0411] R. K. Saiki et al., Science 239, 1988, pp. 487-491.

[0412] Morinaga et al., 1984, Biotechnology 2, pp. 646-639.

[0413] Nelson and Long, Analytical Biochemistry 180, 1989, pp.147-151.

[0414] Hunkapiller et al., 1984, Nature 310, pp. 105-111.

[0415] R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general methodof in vitro preparation and specific mutagenesis of DNA fragments: studyof protein and DNA interactions. Nucl. Acids Res. 16, pp. 7351-7367.

[0416] Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.

[0417] Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.

[0418] S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.

[0419] Boel et al., 1990, Biochemistry 29, pp. 6244-6249.

[0420] Deng and Nickoloff, 1992, Anal. Biochem. 200, pp. 81-88.

1 32 485 amino acids amino acid single linear peptide 1 His His Asn GlyThr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro AsnAsp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu LysSer Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly ThrSer Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu GlyGlu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr ArgAsn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile GlnVal Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 GlyThr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln AsnLys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp GluVal Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala AspVal Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg Asn TrpGly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg IleAsp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp TrpLeu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala ValAla Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr LeuAsn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu HisTyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp MetArg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro ThrHis Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly GluAla Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 TyrAla Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr ArgGlu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile MetSer Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys AsnLys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr GlyThr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val AsnGly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 485 aminoacids amino acid single linear peptide 2 His His Asn Gly Thr Asn Gly ThrMet Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn HisTrp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly IleThr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn AspVal Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn GlnLys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu GluSer Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly AspVal Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn ValLeu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile SerGly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro GlyArg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 HisPhe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val LysHis 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His ValArg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe TrpLys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr AsnTrp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu TyrAsn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu LeuAsn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val ThrPhe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu SerPhe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile LeuThr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr TyrGly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile AspPro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 GlnHis Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser ValSer 465 470 475 480 Ile Trp Val Lys Arg 485 514 amino acids amino acidsingle linear peptide 3 Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr PheGlu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val AlaAsn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp LeuPro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly ValTyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Ala Val ArgThr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln AlaAla His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp HisLys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu ValAsn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln IleGln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr TyrSer Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val AspTrp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg GlyIle Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly AsnTyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro GluVal Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn 210 215 220 ThrThr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Asp Val Arg Ser Gln Thr Gly 245250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys260 265 270 Leu His Asn Tyr Ile Met Lys Thr Asn Gly Thr Met Ser Leu PheAsp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser GlyGly Thr 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met LysAsp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His AspThr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp PheLys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly TyrPro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr AsnIle Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala ArgArg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp HisSer Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu Lys ProGly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly SerLys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe TyrAsp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 AspGly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Trp Ser Ile Thr Thr 485490 495 Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val500 505 510 Ala Trp 1455 base pairs nucleic acid single linear DNA(genomic) 4 CATCATAATG GAACAAATGG TACTATGATG CAATATTTCG AATGGTATTTGCCAAATGAC 60 GGGAATCATT GGAACAGGTT GAGGGATGAC GCAGCTAACT TAAAGAGTAAAGGGATAACA 120 GCTGTATGGA TCCCACCTGC ATGGAAGGGG ACTTCCCAGA ATGATGTAGGTTATGGAGCC 180 TATGATTTAT ATGATCTTGG AGAGTTTAAC CAGAAGGGGA CGGTTCGTACAAAATATGGA 240 ACACGCAACC AGCTACAGGC TGCGGTGACC TCTTTAAAAA ATAACGGCATTCAGGTATAT 300 GGTGATGTCG TCATGAATCA TAAAGGTGGA GCAGATGGTA CGGAAATTGTAAATGCGGTA 360 GAAGTGAATC GGAGCAACCG AAACCAGGAA ACCTCAGGAG AGTATGCAATAGAAGCGTGG 420 ACAAAGTTTG ATTTTCCTGG AAGAGGAAAT AACCATTCCA GCTTTAAGTGGCGCTGGTAT 480 CATTTTGATG GGACAGATTG GGATCAGTCA CGCCAGCTTC AAAACAAAATATATAAATTC 540 AGGGGAACAG GCAAGGCCTG GGACTGGGAA GTCGATACAG AGAATGGCAACTATGACTAT 600 CTTATGTATG CAGACGTGGA TATGGATCAC CCAGAAGTAA TACATGAACTTAGAAACTGG 660 GGAGTGTGGT ATACGAATAC ACTGAACCTT GATGGATTTA GAATAGATGCAGTGAAACAT 720 ATAAAATATA GCTTTACGAG AGATTGGCTT ACACATGTGC GTAACACCACAGGTAAACCA 780 ATGTTTGCAG TGGCTGAGTT TTGGAAAAAT GACCTTGGTG CAATTGAAAACTATTTGAAT 840 AAAACAAGTT GGAATCACTC GGTGTTTGAT GTTCCTCTCC ACTATAATTTGTACAATGCA 900 TCTAATAGCG GTGGTTATTA TGATATGAGA AATATTTTAA ATGGTTCTGTGGTGCAAAAA 960 CATCCAACAC ATGCCGTTAC TTTTGTTGAT AACCATGATT CTCAGCCCGGGGAAGCATTG 1020 GAATCCTTTG TTCAACAATG GTTTAAACCA CTTGCATATG CATTGGTTCTGACAAGGGAA 1080 CAAGGTTATC CTTCCGTATT TTATGGGGAT TACTACGGTA TCCCAACCCATGGTGTTCCG 1140 GCTATGAAAT CTAAAATAGA CCCTCTTCTG CAGGCACGTC AAACTTTTGCCTATGGTACG 1200 CAGCATGATT ACTTTGATCA TCATGATATT ATCGGTTGGA CAAGAGAGGGAAATAGCTCC 1260 CATCCAAATT CAGGCCTTGC CACCATTATG TCAGATGGTC CAGGTGGTAACAAATGGATG 1320 TATGTGGGGA AAAATAAAGC GGGACAAGTT TGGAGAGATA TTACCGGAAATAGGACAGGC 1380 ACCGTCACAA TTAATGCAGA CGGATGGGGT AATTTCTCTG TTAATGGAGGGTCCGTTTCG 1440 GTTTGGGTGA AGCAA 1455 1455 base pairs nucleic acidsingle linear DNA (genomic) 5 CATCATAATG GGACAAATGG GACGATGATGCAATACTTTG AATGGCACTT GCCTAATGAT 60 GGGAATCACT GGAATAGATT AAGAGATGATGCTAGTAATC TAAGAAATAG AGGTATAACC 120 GCTATTTGGA TTCCGCCTGC CTGGAAAGGGACTTCGCAAA ATGATGTGGG GTATGGAGCC 180 TATGATCTTT ATGATTTAGG GGAATTTAATCAAAAGGGGA CGGTTCGTAC TAAGTATGGG 240 ACACGTAGTC AATTGGAGTC TGCCATCCATGCTTTAAAGA ATAATGGCGT TCAAGTTTAT 300 GGGGATGTAG TGATGAACCA TAAAGGAGGAGCTGATGCTA CAGAAAACGT TCTTGCTGTC 360 GAGGTGAATC CAAATAACCG GAATCAAGAAATATCTGGGG ACTACACAAT TGAGGCTTGG 420 ACTAAGTTTG ATTTTCCAGG GAGGGGTAATACATACTCAG ACTTTAAATG GCGTTGGTAT 480 CATTTCGATG GTGTAGATTG GGATCAATCACGACAATTCC AAAATCGTAT CTACAAATTC 540 CGAGGTGATG GTAAGGCATG GGATTGGGAAGTAGATTCGG AAAATGGAAA TTATGATTAT 600 TTAATGTATG CAGATGTAGA TATGGATCATCCGGAGGTAG TAAATGAGCT TAGAAGATGG 660 GGAGAATGGT ATACAAATAC ATTAAATCTTGATGGATTTA GGATCGATGC GGTGAAGCAT 720 ATTAAATATA GCTTTACACG TGATTGGTTGACCCATGTAA GAAACGCAAC GGGAAAAGAA 780 ATGTTTGCTG TTGCTGAATT TTGGAAAAATGATTTAGGTG CCTTGGAGAA CTATTTAAAT 840 AAAACAAACT GGAATCATTC TGTCTTTGATGTCCCCCTTC ATTATAATCT TTATAACGCG 900 TCAAATAGTG GAGGCAACTA TGACATGGCAAAACTTCTTA ATGGAACGGT TGTTCAAAAG 960 CATCCAATGC ATGCCGTAAC TTTTGTGGATAATCACGATT CTCAACCTGG GGAATCATTA 1020 GAATCATTTG TACAAGAATG GTTTAAGCCACTTGCTTATG CGCTTATTTT AACAAGAGAA 1080 CAAGGCTATC CCTCTGTCTT CTATGGTGACTACTATGGAA TTCCAACACA TAGTGTCCCA 1140 GCAATGAAAG CCAAGATTGA TCCAATCTTAGAGGCGCGTC AAAATTTTGC ATATGGAACA 1200 CAACATGATT ATTTTGACCA TCATAATATAATCGGATGGA CACGTGAAGG AAATACCACG 1260 CATCCCAATT CAGGACTTGC GACTATCATGTCGGATGGGC CAGGGGGAGA GAAATGGATG 1320 TACGTAGGGC AAAATAAAGC AGGTCAAGTTTGGCATGACA TAACTGGAAA TAAACCAGGA 1380 ACAGTTACGA TCAATGCAGA TGGATGGGCTAATTTTTCAG TAAATGGAGG ATCTGTTTCC 1440 ATTTGGGTGA AACGA 1455 1548 basepairs nucleic acid single linear DNA (genomic) 6 GCCGCACCGT TTAACGGCACCATGATGCAG TATTTTGAAT GGTACTTGCC GGATGATGGC 60 ACGTTATGGA CCAAAGTGGCCAATGAAGCC AACAACTTAT CCAGCCTTGG CATCACCGCT 120 CTTTGGCTGC CGCCCGCTTACAAAGGAACA AGCCGCAGCG ACGTAGGGTA CGGAGTATAC 180 GACTTGTATG ACCTCGGCGAATTCAATCAA AAAGGGACCG TCCGCACAAA ATACGGAACA 240 AAAGCTCAAT ATCTTCAAGCCATTCAAGCC GCCCACGCCG CTGGAATGCA AGTGTACGCC 300 GATGTCGTGT TCGACCATAAAGGCGGCGCT GACGGCACGG AATGGGTGGA CGCCGTCGAA 360 GTCAATCCGT CCGACCGCAACCAAGAAATC TCGGGCACCT ATCAAATCCA AGCATGGACG 420 AAATTTGATT TTCCCGGGCGGGGCAACACC TACTCCAGCT TTAAGTGGCG CTGGTACCAT 480 TTTGACGGCG TTGATTGGGACGAAAGCCGA AAATTGAGCC GCATTTACAA ATTCCGCGGC 540 ATCGGCAAAG CGTGGGATTGGGAAGTAGAC ACGGAAAACG GAAACTATGA CTACTTAATG 600 TATGCCGACC TTGATATGGATCATCCCGAA GTCGTGACCG AGCTGAAAAA CTGGGGGAAA 660 TGGTATGTCA ACACAACGAACATTGATGGG TTCCGGCTTG ATGCCGTCAA GCATATTAAG 720 TTCAGTTTTT TTCCTGATTGGTTGTCGTAT GTGCGTTCTC AGACTGGCAA GCCGCTATTT 780 ACCGTCGGGG AATATTGGAGCTATGACATC AACAAGTTGC ACAATTACAT TACGAAAACA 840 GACGGAACGA TGTCTTTGTTTGATGCCCCG TTACACAACA AATTTTATAC CGCTTCCAAA 900 TCAGGGGGCG CATTTGATATGCGCACGTTA ATGACCAATA CTCTCATGAA AGATCAACCG 960 ACATTGGCCG TCACCTTCGTTGATAATCAT GACACCGAAC CCGGCCAAGC GCTGCAGTCA 1020 TGGGTCGACC CATGGTTCAAACCGTTGGCT TACGCCTTTA TTCTAACTCG GCAGGAAGGA 1080 TACCCGTGCG TCTTTTATGGTGACTATTAT GGCATTCCAC AATATAACAT TCCTTCGCTG 1140 AAAAGCAAAA TCGATCCGCTCCTCATCGCG CGCAGGGATT ATGCTTACGG AACGCAACAT 1200 GATTATCTTG ATCACTCCGACATCATCGGG TGGACAAGGG AAGGGGGCAC TGAAAAACCA 1260 GGATCCGGAC TGGCCGCACTGATCACCGAT GGGCCGGGAG GAAGCAAATG GATGTACGTT 1320 GGCAAACAAC ACGCTGGAAAAGTGTTCTAT GACCTTACCG GCAACCGGAG TGACACCGTC 1380 ACCATCAACA GTGATGGATGGGGGGAATTC AAAGTCAATG GCGGTTCGGT TTCGGTTTGG 1440 GTTCCTAGAA AAACGACCGTTTCTACCATC GCTCGGCCGA TCACAACCCG ACCGTGGACT 1500 GGTGAATTCG TCCGTTGGACCGAACCACGG TTGGTGGCAT GGCCTTGA 1548 485 amino acids amino acid singlelinear peptide 7 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe GluTrp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn SerAsp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile ProPro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala TyrAsp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg ThrLys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser LeuLys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His LysGly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val AsnPro Asn Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile GluAla Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His SerSer Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp TrpAsp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg GlyHis Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly AsnTyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro GluVal Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn ThrLeu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 IleLys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val GlnArg 305 310 315 320 His Pro Ser His Ala Val Thr Phe Val Asp Asn His AspSer Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp PheLys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly TyrPro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His GlyVal Pro Ala Met Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala ArgGln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp HisHis Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His ProAsn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly SerLys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp SerAsp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala AspGly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 IleTrp Val Asn Lys 485 30 base pairs nucleic acid single linear 8GCTGCGGTGA CCTCTTTAAA AAATAACGGC 30 24 base pairs nucleic acid singlelinear 9 CCACCGCTAT TAGATGCATT GTAC 24 32 base pairs nucleic acid singlelinear 10 CTTACGTATG CAGACGTCGA TATGGATCAC CC 32 34 base pairs nucleicacid single linear 11 GATCCATATC GACGTCTGCA TACGTAAGAT AGTC 34 23 basepairs nucleic acid single linear 12 TTASGGGCAA GGCCTGGGAC TGG 23 37 basepairs nucleic acid single linear 13 CCCAGGCCTT GCCCSTAAAT TTATATATTTTGTTTTG 37 31 base pairs nucleic acid single linear 14 GGTTTCGGTTCGAAGGATTC ACTTCTACCG C 31 33 base pairs nucleic acid single linear 15GCGGTAGAAG TGAATCCTTC GAACCGAAAC CAG 33 43 base pairs nucleic acidsingle linear 16 GGTACTATCG TAACAATGGC CGATTGCTGA CGCTGTTATT TGC 43 28base pairs nucleic acid single linear 17 CTGTGACTGG TGAGTACTCA ACCAAGTC28 35 base pairs nucleic acid single linear 18 CTACTTCCCA ATCCCAAGCTTTACCTCGGA ATTTG 35 35 base pairs nucleic acid single linear 19CAAATTCCGA GGTAAAGCTT GGGATTGGGA AGTAG 35 24 base pairs nucleic acidsingle linear 20 TTGAACAACC GTTCCATTAA GAAG 24 60 base pairs nucleicacid single linear 21 CTCTGTATCG ACTTCCCAGT CCCAAGCTTT TGTCCTGAATTTATATATTT TGTTTTGAAG 60 60 base pairs nucleic acid single linear 22CTCTGTATCG ACTTCCCAGT CCCAAGCTTT GCCTCCGAAT TTATATATTT TGTTTTGAAG 60 51base pairs nucleic acid single linear 23 ATGTGTAAGC CAATCGCGAGTAAAGCTAAA TTTTATATGT TTCACTGCAT C 51 34 base pairs nucleic acid singlelinear 24 GCACCAAGGT CATTTCGCCA GAATTCAGCC ACTG 34 39 base pairs nucleicacid single linear 25 TGTCAGAACC AACGCGTATG CACATGGTTT AAACCATTG 39 42base pairs nucleic acid single linear 26 ACCACCTGGA CCATCGCTGCAGATGGTGGC AAGGCCTGAA TT 42 36 base pairs nucleic acid single linear 27GGCAAAAGTT TGACGTGCCT CGAGAAGAGG GTCTAT 36 36 base pairs nucleic acidsingle linear 28 TTGTCCCGCT TTATTCTGGC CAACATACAT CCATTT 36 37 basepairs nucleic acid single linear 29 CCCAATCCCA AGCTTTACCA YCGAACTTGTAGATACG 37 37 base pairs nucleic acid single linear 30 CCCAATCCCAAGCTTTATCT CSGAACTTGT AGATACG 37 34 base pairs nucleic acid singlelinear 31 GATCCATATC GACGTCTGCA TACAGTAAAT AATC 34 34 base pairs nucleicacid single linear cDNA 32 GATCCATATC GACGTCTGCA TAAATTAAAT AATC 34

1-29. (Canceled).
 30. A method for producing ethanol from starch,comprising: a) treating starch with an alpha-amylase, wherein thealpha-amylase is selected from the group consisting of: an alpha-amylasecomprising an amino acid sequence having at least 80% homology to SEQ IDNO:1 and which alpha-amylase is modified by having an amino aciddeletion of two amino acids selected from the group of amino acidsequivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:1;an alpha-amylase comprising an amino acid sequence having at least 80%homology to SEQ ID NO:2 and which alpha-amylase is modified by having anamino acid deletion of two amino acids selected from the group of aminoacids equivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ IDNO:2; an alpha-amylase comprising an amino acid sequence having at least80% homology to SEQ ID NO:3 and which alpha-amylase is modified byhaving an amino acid deletion of two amino acids selected from the groupof amino acids equivalent to positions 178, 179, 180, 181, 182 and 183in SEQ ID NO:3; and an alpha-amylase comprising an amino acid sequencehaving at least 80% homology to SEQ ID NO:7 and which alpha-amylase ismodified by having an amino acid deletion of two amino acids selectedfrom the group of amino acids equivalent to positions 180, 181, 182,183, 184 and 185 in SEQ ID NO:7; and b) preparing ethanol from thetreated starch.
 31. The method of claim 30, wherein said methodcomprises treating starch with an alpha-amylase that comprises an aminoacid sequence having at least 80% homology to SEQ ID NO:1 and whichalpha-amylase is modified by having an amino acid deletion of two aminoacids selected from the group of amino acids equivalent to positions180, 181, 182, 183, 185 and 185 in SEQ ID NO:1.
 32. The method of claim31, wherein the alpha amylase comprises a deletion of three amino acidsselected from the group of amino acids equivalent to positions 180, 181,182, 183, 185 and 185 in SEQ ID NO:1.
 33. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 182 in SEQ ID NO.
 1. 34. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 183 and 184 in SEQ ID NO.
 1. 35. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 183 in SEQ ID NO.
 1. 36. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 182 and 183 in SEQ ID NO.
 1. 37. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 182 and 184 in SEQ ID NO.
 1. 38. The method of claim 31,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 184 in SEQ ID NO.
 1. 39. The method of claim 31,wherein the alpha-amylase comprises an amino acid sequence having atleast 85% homology to SEQ ID NO:1.
 40. The method of claim 31, whereinthe alpha-amylase comprises an amino acid sequence having at least 90%homology to SEQ ID NO:1.
 41. The method of claim 31, wherein thealpha-amylase comprises an amino acid sequence having at least 95%homology to SEQ ID NO:
 1. 42. The method of claim 30, wherein saidmethod comprises treating starch with an alpha-amylase that comprises anamino acid sequence having at least 80% homology to SEQ ID NO:2 andwhich alpha-amylase is modified by having an amino acid deletion of twoamino acids selected from the group of amino acids equivalent topositions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:2.
 43. The methodof claim 42, wherein the alpha amylase comprises a deletion of threeamino acids selected from the group of amino acids equivalent topositions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:2.
 44. The methodof claim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 181 and 182 in SEQ ID NO.
 2. 45. The method ofclaim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 183 and 184 in SEQ ID NO.
 2. 46. The method ofclaim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 181 and 183 in SEQ ID NO.
 2. 47. The method ofclaim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 182 and 183 in SEQ ID NO.
 2. 48. The method ofclaim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 182 and 184 in SEQ ID NO.
 2. 49. The method ofclaim 42, wherein the alpha-amylase comprises a deletion at positionsequivalent to positions 181 and 184 in SEQ ID NO.
 2. 50. The method ofclaim 42, wherein the alpha-amylase comprising an amino acid sequencehaving at least 85% homology to SEQ ID NO:2.
 51. The method of claim 42,wherein the alpha-amylase comprising an amino acid sequence having atleast 90% homology to SEQ ID NO:2.
 52. The method of claim 42, whereinthe alpha-amylase comprising an amino acid sequence having at least 95%homology to SEQ ID NO:2.
 53. The method of claim 30, wherein said methodcomprises treating starch with an alpha-amylase that comprises an aminoacid sequence having at least 80% homology to SEQ ID NO:3 and whichalpha-amylase is modified by having an amino acid deletion of two aminoacids selected from the group of amino acids equivalent to positions178, 179, 180, 181, 182 and 183 in SEQ ID NO:3.
 54. The method of claim53, wherein the alpha amylase comprises a deletion of three amino acidsselected from the group of amino acids equivalent to positions 178, 179,180, 181, 182 and 183 in SEQ ID NO:3.
 55. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 179 and 180 in SEQ ID NO.
 3. 56. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 182 in SEQ ID NO.
 3. 57. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 179 and 181 in SEQ ID NO.
 3. 58. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 180 and 181 in SEQ ID NO.
 3. 59. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 180 and 182 in SEQ ID NO.
 3. 60. The method of claim 53,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 179 and 182 in SEQ ID NO.
 3. 61. The method of claim 53,wherein the alpha-amylase comprises an amino acid sequence having atleast 85% homology to SEQ ID NO:3.
 62. The method of claim 53, whereinthe alpha-amylase comprises an amino acid sequence having at least 90%homology to SEQ ID NO:3.
 63. The method of claim 53, wherein thealpha-amylase comprises an amino acid sequence having at least 95%homology to SEQ ID NO:3.
 64. The method of claim 30, wherein said methodcomprises treating starch with an alpha-amylase that comprises an aminoacid sequence having at least 80% homology to SEQ ID NO:7 and whichalpha-amylase is modified by having a deletion of two amino acidsselected from the group of amino acids equivalent to positions 180, 181,182, 183, 184 and 185 in SEQ ID NO:7.
 65. The method of claim 64,wherein the alpha amylase comprises a deletion of three amino acidsselected from the group of amino acids equivalent to positions 180, 181,182, 183, 184 and 185 in SEQ ID NO:7.
 66. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 182 in SEQ ID NO.
 7. 67. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 183 and 184 in SEQ ID NO.
 7. 68. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 183 in SEQ ID NO.
 7. 69. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 182 and 183 in SEQ ID NO.
 7. 70. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 182 and 184 in SEQ ID NO.
 7. 71. The method of claim 64,wherein the alpha-amylase comprises a deletion at positions equivalentto positions 181 and 184 in SEQ ID NO.
 7. 72. The method of claim 64,wherein the alpha-amylase comprises an amino acid sequence having atleast 85% homology to SEQ ID NO:7.
 73. The method of claim 64, whereinthe alpha-amylase comprises an amino acid sequence having at least 90%homology to SEQ ID NO:7.
 74. The method of claim 64, wherein thealpha-amylase comprises an amino acid sequence having at least 95%homology to SEQ ID NO:7.
 75. A method for producing a sweetener fromstarch, comprising: a) treating starch with an alpha-amylase, whereinthe alpha-amylase is selected from the group consisting of: analpha-amylase comprising an amino acid sequence having at least 80%homology to SEQ ID NO:1 and which alpha-amylase is modified by having adeletion of two amino acids selected from the group of amino acidsequivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:1;an alpha-amylase comprising an amino acid sequence having at least 80%homology to SEQ ID NO:2 and which alpha-amylase is modified by having adeletion of two amino acids selected from the group of amino acidsequivalent to positions 180, 181, 182, 183, 185 and 185 in SEQ ID NO:2;an alpha-amylase comprising an amino acid sequence having at least 80%homology to SEQ ID NO:3 and which alpha-amylase is modified by having adeletion of two amino acids selected from the group of amino acidsequivalent to positions 178, 179, 180, 181, 182 and 183 in SEQ ID NO:3;and an alpha-amylase comprising an amino acid sequence having at least80% homology to SEQ ID NO:7 and which alpha-amylase is modified byhaving a deletion of two amino acids selected of an amino acid selectedfrom the group of amino acids equivalent to positions 180, 181, 182,183, 184 and 185 in SEQ ID NO:7; and b) preparing the sweetener from thetreated starch.